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Farnesol Signaling in Candida albicans
Melanie L. Langford
University of Nebraska at Lincoln, email@example.com
Langford, Melanie L., "Farnesol Signaling in Candida albicans" (2010). Dissertations and Theses in Biological Sciences. Paper 9. http://digitalcommons.unl.edu/bioscidiss/9
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Farnesol Signaling in Candida albicans
Melanie L. Langford
Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy
Major: Biological Sciences
Under the Supervision of Professor Audrey L. Atkin
Farnesol Signaling in Candida albicans Melanie L. Langford, Ph.D. University of Nebraska, 2010
Advisor: Audrey L. Atkin Candida albicans is a polymorphic fungus that causes a range of disease in humans, from mucosal infections to systemic disease. Its ability to cause disease is linked to conversion between yeast and filamentous forms of growth, and the first quorum-sensing molecule discovered in an eukaryote, farnesol, blocks this transition. In C. albicans, farnesol also kills mating-competent opaque cells, inhibits biofilm formation, protects the cells from oxidative stress, and can be a virulence factor or protective agent in disseminated and mucosal mouse models of infection, respectively. While much emphasis has been placed on determining its effect on C. albicans morphology, the molecular response to farnesol is not completely understood. The overall theme for this dissertation was to better understand the C. albicans molecular response to farnesol under quorum sensing conditions. Due to the duplicitous nature of the farnesol response in C. albicans, i.e., its ability to kill cells or simply alter morphology, we clearly defined the environmental conditions in which farnesol acts as a quorum sensing molecule or as a toxic agent towards C. albicans. This clarification enabled a subsequent two-pronged approach to study the molecular response to farnesol during morphological regulation. A direct approach was used to investigate the role of a likely candidate, Tup1, a negative regulator of hyphal development, in farnesol signaling. Secondly, a screening approach was utilized to identify new farnesol resistant mutants that may participate in the farnesol
From the mutants identified. albicans zinc finger) was selected for further characterization and was shown to play a vital role in the morphological response to farnesol as well as farnesol tolerance. . and highlights the power of farnesol as a tool with which to unravel the complex signaling networks present in C. this study identified two new factors involved in farnesol signaling.response. albicans. Overall. Czf1 (C.
During my time spent in your lab. what initially seems like more work always pays off in learning experiences.iv ACKNOWLEDGEMENTS No one ever gets through their Ph. and I am certainly no exception. I am happy you encouraged me to give several oral and poster presentations. I also think it says a lot about the career development information you provide your lab members when I have heard nearly everything discussed in our Professionalism course this semester from you already over the course of my graduate program. I would like to begin by thanking my wonderful advisor. personalities. I have truly enjoyed having joint Atkin-Nickerson lab meetings. or personal). To both Audrey and Ken. I will miss having a chemistry expert right next door and your enthusiasm for nearly everything and anything in science.D. I would also like to extend my thanks to Kenneth Nickerson. I think I have . I am always amazed at how well your collaboration works (given your entirely different research styles. my favorite fungal biology neighbor. program on their own. Lastly I always appreciated your support whenever a crisis arose (be it professional. and I have had the chance to learn several new lab techniques as well. experimental. and I feel they have been critical to the completion of my degree. What a happy accident joining your lab turned out to be. Audrey Atkin. but I am happy that it does (maybe I fall somewhere in the middle of you two?). such as working with RNA. things that I will miss. etc). I have learned nearly everything I know about fungi. both locally and nationally.I never would have guessed that I would switch from studying bacteriology to fungal biology! It makes an incredible difference to work for someone that not only encourages quality research but is so pleasant on a personal level as well.
I would like to thank Bessie Kebaara. Krista Fager. Of the current students in our research group. for being such an excellent collaborator on the farnesol toxicity project. and even though things took longer to finish than we originally thought (seems they always do).D. I would first like to extend my gratitude to Sahar Hasim. You always provided interesting ideas and suggestions during my committee meetings. I could always go to you with technical questions. but not so easy to find someone to teach you the method and theory behind it. and I have also enjoyed writing papers with both of you and learning about the process of paper submission. or just to complain about the cold weather. Next I would like to thank all my committee members: Steve Harris. both taught me important Candida techniques and were great role models for me as a new student. and I appreciate that you always did so in a very constructive manner. Jack Morris. First. and Luwen Zhang.it is easy to find people that will let you borrow their machines. student. There are also so many past and current lab members to thank. students in the Nickerson lab. former Ph.v learned about balancing careful experimental design with exploratory experiments moving in new directions. for teaching me farnesol extraction techniques and for being a good friend. Jeff Bunker. I think we learned some very valuable information over the course of that project.D. a former post-doc in the lab that I still miss having around. Dhammika Navarathna and Raluca Dumitru. Thanks to Cheryl Bailey for teaching me real-time PCR from scratch. And thanks to Suman Ghosh. scientific ideas. and Narmin Tahirova for all your helpful comments and suggestions during journal club and lab meetings. another former Ph. Thanks also to Swetha Tati (my technical support aide prior to my defense). You are such an amiable and reliable person to work with. .
Believe it or not. Dad. and Ph. It has been chaotic but fun to go through the ups and downs of graduate school with you. Courtney. And lastly. Gina.D. and all the Langford brothers. as you were always the perfect confidant. I am certain that I would not have been able to get through graduate school without you. degrees. I would like to thank you for all of your support during both my M.S. Jim.D. Gabe. I would like to thank my husband.vi To my family: Mom. degree. . I am so excited to start the next phase of our life together with our new family. much less have the confidence to even pursue my Ph. attempting to explain to you what I do in the lab has actually improved my oral presentations to more general scientific audiences! I am very lucky to have such supportive family members on my side.
...................................................................................................................................................................................................................................................................................................49 Figures...............................................................................................................................................50 .....36 Introduction..................................................................................................................................................x LIST OF TABLES .....................................................................................39 Results.........................................................................................1 References.................................vii TABLE OF CONTENTS ACKNOWLEDGEMENTS.....18 Figures...........................................................................................................42 References.....................................................................................40 Discussion ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................... ................... xi CHAPTER 1: Introduction...........23 Figure Legends.............................37 Methods.........44 Figure Legends..................................................................20 CHAPTER 2: Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions.................................................................................................................................................... vii LIST OF FIGURES .............................................. iv TABLE OF CONTENTS .............................................................19 Tables...............................................................................................................35 Abstract ......................................................................................................................
............................................130 .........90 Introduction.................................................................................................................................91 Methods....................................................................................................................................................................................................................................................................119 Figures......................................................................................................................54 Introduction..............................................................................................................................................................................................................................................................................................................................94 Results...............................................................................................................106 References......................................................................................53 Abstract ..................................................................73 Figure Legends.........59 Results......................55 Methods............................68 References.................................................................................................................................................................................................................................................................................................................................................................................................................................................................................80 Figures.........................................89 Abstract ....................................................................................................................62 Discussion ........................................................................................................122 Tables.............................................................................111 Figure Legends..............87 CHAPTER 4: A novel role for Czf1 in farnesol tolerance and the morphological response to farnesol in Candida albicans ..........................97 Discussion ...............................................................................................................................................................................................................................................................................................82 Tables..............................................viii CHAPTER 3: Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous growth induction .......
...................157 .................ix CHAPTER 5: Summary and Future Directions .............................147 References................................................................................................................
........................................................x LIST OF FIGURES Figure 1-1.................84 Figure 3-4..........................................................................................85 Figure 3-5........86 Figure 4-1..........................................127 Figure 4-6......................................................129 .........................................................................................................................................................................................................................124 Figure 4-4..............................................................................................................................................................................................122 Figure 4-2........123 Figure 4-3..............128 Figure 4-7...................................19 Figure 2-1................................................................................................................................................................................50 Figure 2-2...............................................................................................................................51 Figure 2-3............................................................................................................................................................................................................................................................................82 Figure 3-2..........83 Figure 3-3................................................................................ 125-126 Figure 4-5....................................................................................................................................................................52 Figure 3-1.......................................................................................................................................................................................................................................................................................................................................................................................................
............................................................................................................................130 Table 4-2 ......................................................................xi LIST OF TABLES Table 1-1................................................... ............................................................................................................................................ 20-21 Table 1-2 .................................................131 Supplementary Table 4-1........................87 Table 3-2 ....................................22 Table 3-1 ..........................................................................................................88 Table 4-1 ....................................................................................................................... 132-146 .........................................................................................................
Future Microbiol. and Nickerson.L.. K.1 CHAPTER 1 INTRODUCTION Reference: Langford. . a Quorum Sensing Molecule Produced by Candida albicans.L. M. A.W. Atkin.. (2009) Cellular Interactions of Farnesol. 4: 1353-62.
in aerobic conditions the cells die by necrosis (Dumitru et al. 2005). Oh et al. can be adversely affected by farnesol.. Lastly. albicans is a polymorphic fungus that is capable of growing in yeast. The mating-competent form of C. Given that it may affect all five C. a conversion that is the focus of intense study in C. 2001).. like farnesol. 2005.E-farnesol as a quorum sensing molecule in Candida albicans (Hornby et al.. 2001). The earlier work on the discovery and characterization of farnesol was reviewed by Nickerson et al (Nickerson et al..2 INTRODUCTION The discovery of E. acts to block the yeast to filament transition (Oh et al. 2007. Farnesoic acid. but it is far less active than farnesol (Shchepin et al. 2007) but remain unharmed in anaerobic conditions (Dumitru et al... Saville et al. a C.. opaque. Another quorum sensing molecule (QSM) described in C... a process once thought to be confined to bacteria.. 2006. 2001). 2001). 2001) demonstrated the existence of quorum sensing in a eukaryotic organism.. 2009). 2003).. 10231 (Hornby and Nickerson. albicans cell morphology with unknown function.. is increased in the presence of very high (10 mM) levels of farnesol (Martin et al.. albicans growth morphologies. Additional possible quorum sensing molecules are reviewed by Kruppa (Kruppa. opaque cells. 2002) in a manner consistent with its inhibition of the yeast to filament conversion... it has . albicans (Alem et al. albicans is farnesoic acid (Oh et al. Farnesol also blocks biofilm formation by C. 1997. Farnesol is capable of blocking the yeast to hyphal/pseudohyphal (filaments) switch (Hornby et al. hyphal. Ramage et al. 2003) and is produced by only one known strain of C. the production of chlamydospores. Martins et al. 2004. albicans because of its role in virulence (Lo et al. albicans. pseudohyphal. 2006). 2004). and chlamydospore cell morphologies. albicans. C. Cao et al.
histone genes. 2007). albicans farnesol response. 2008). 2008). and mitogen activated protein (MAP) kinase pathway components. Enjalbert and Whiteway. The presence of farnesol increases TUP1 expression and suppresses the haploinsufficient phenotype of a TUP1/tup1Δ mutant (Kebaara et al. cyclin and cell proliferation genes. 2001. as discussed later. 2008). phagocytosis response genes. Cho et al. Tup1 and Nrg1. and several factors involved in the filamentation process are known to play a role in the C. as tup1Δ/tup1Δ and nrg1Δ/nrg1Δ mutants are unable to respond to farnesol (Kebaara et al. 2004). but Cph1 might play a downstream role since a cph1Δ/cph1Δ mutant retains the ability to respond to farnesol (Davis-Hanna et al. may also be involved in the response to farnesol. drug resistance genes. 2004). 2005. The Molecular Response to Farnesol in C. Ras1 of the cyclic AMP pathway... 2005.. it promotes the question: Does farnesol have one or more specific target(s) in . is a candidate for direct inhibition by farnesol (Davis-Hanna et al... and the search is underway to elucidate the molecular mechanism(s) of these effects.. Microarray analyses have identified other categories of genes affected by farnesol treatment such as heat shock genes.. 2008). Uppuluri et al.. Regulators of filamentation are not the only genes involved in the farnesol response. and adhesion genes (Cao et al.. also play critical roles in the farnesol response.. Repressors of filamentation. 2007. Cph1 and Hst1 (Sato et al. genes expressed at high cell density. albicans The first described function of farnesol was to block filamentation induced by a variety of environmental signals (Hornby et al. Chk1 (Kruppa et al.. Mosel et al. albicans physiology. With all of the different pathways and responses induced by farnesol. 2005). A two-component signal transduction pathway histidine kinase.3 become clear that farnesol plays an influential role in C.
E-farnesol. but not the sole explanation for these results is the presence of a farnesol receptor(s) or farnesol binding protein(s) in C.. providing support for the specific target(s) model. 2005) and a .. there are four isomers of farnesol but only E.E-farnesol is capable of blocking filamentation in C. Other farnesol analogs and related molecules from other organisms. 2005). Shchepin et al. suggesting some limited flexibility for the inhibitory action of farnesol-related molecules (Davis-Hanna et al. respectively (Shchepin et al. This conclusion is based on a comparison between pure (96%) E. 2003). 2003. are capable of blocking filamentation in C. Fluorescent farnesol (Shchepin et al.4 µM. rather than a scenario involving general membrane disruption.E-farnesol and two commercial mixed isomers containing 56 and 36% E.. Thus. 3. or does it somehow elicit a more general and nonspecific response with pleomorphic consequences? Are there farnesol receptors or farnesol binding proteins? The morphological response to farnesol in C. there was sufficient E.5. albicans. 2004... 2005)..4 the cell.. Kim et al. These three farnesol preparations reduced germ tube formation (early stage filaments) to 50% at 1. albicans appears to be very sensitive to minor changes in the structure of farnesol. Kim et al. 2004. 2002. albicans.2. such as 3-oxo-C12-homoserine lactone and dodecanol.. 2008.E-farnesol in the two samples with mixed isomers to account for all of their QSM activities. 2002. Further. Hogan et al. and 4. albicans (Shchepin et al. 2003... Shchepin et al. One. For example. 2003). Shchepin et al. the 12-carbon backbone of farnesol appears to be critical for its ability to block filamentation because altering the chain length generally results in decreased filament inhibitory activity (Hogan et al.. Shchepin et al..
and it has been estimated that the farnesylated. see review by Hancock et al (Hancock. 1) have been proposed as a potential direct target for farnesol inhibition (Davis-Hanna et al. albicans.. 1). 1998. 2003). releasing farnesylated Ras1 from the membrane (Fig. 1B). where measured. It is unlikely that farnesylated Ras1 has the farnesyl tail removed because it is attached by a very stable thioether bond and. discussed in more detail by Paula Sundstrom and Deborah Hogan in this issue). 2006) were developed to help isolate farnesol binding proteins. so far with limited success. albicans and farnesol inhibition of filament formation was itself reversed by addition of dibutyrylcAMP (Davis-Hanna et al. Farnesol and Ras1/cAMP Signaling Ras1 and the adenylyl cyclase-cAMP-protein kinase A (PKA)-Efg1 pathway (Fig. 1C). It may be important that Ras1 is one of the few farnesylated proteins in C..5 farnesol affinity column (Shchepin. farnesylated Ras1 has a long half life. 1993). Feng . Having Ras1 as a direct target for farnesol can provide some unity for many of the filamentation genes involved in the farnesol response since it is a common regulator for the Hst7/Cph1 MAP kinase pathway and the cAMP pathway (Castilla et al. membrane-bound form of Ras1 is ca. A partial list includes: blocking the farnesylation of Ras1. This idea is very reasonable and it is persuasive that farnesol was able to reverse filamentation in the dominant active (CAI4-RaslG13V) mutant of C. and binding directly to a downstream protein such as adenylyl cyclase (Fig. Thus.. 100 X more effective in activating adenylyl cyclase than is the cytoplasmic form (Kuroda et al. binding directly to Ras1 (Fig. 2008.. 1A). 2008). there are several ways by which farnesol could inhibit the Ras1-cAMP-PKA-Efg1 pathway (Fig.
A second possibility for farnesol signaling is the presence of multiple farnesol targets.. 2006) and 13. While it is unknown what factors regulate Tup1/Nrg1 and Chk1. it is .009.. Possible Artifacts We have found several artifacts associated with the cellular response to farnesol. each affecting different aspects of filamentation in response to different inducing stimuli.106 mg/g dry weight of cells.. 1999. arg4Δ/arg4Δ) produce germ tubes faster than their wild type.. 2005). Similarly. the values for extracellular. when examining a mutant for its response to farnesol. intracellular. prototrophic counterparts and 2) as a consequence. the membrane-associated farnesol would constitute 1-2% of the total extractable membrane lipids based on a value of 8.5 mg extractable lipid/g dry weight (Hitchcock et al. it is possible that they are also under the control of Ras1.5 µM intracellular. and membrane-associated farnesol were 0. These values correspond to concentrations of ca. 1986). 2001). and 0. This was done to emphasize that further research is warranted to determine the exact nature of the molecular response to farnesol during yeast to filament inhibition. Caveats which must be considered include: 1) some auxotrophic mutants (his1Δ/his1Δ. 0.. respectively (Navarathna et al. On the one occasion when cellular localization was attempted (Navarathna et al.. while a third possibility is that farnesol interacts with the cell membrane in a very specific manner to induce a pleiotropic signaling response in the rest of the cell. 3 µl/mg dry weight. Leberer et al. with the intracellular calculation based on a yeast cytoplasmic volume of ca.115. 2005). 4 µM extracellular (Nickerson et al.6 et al. It is difficult to say whether farnesol acts from the outside or the inside because it can cross the cytoplasmic membrane by diffusion. Notice that Figure 1 has been drawn with both intracellular and extracellular farnesol.
1995) or interference with phosphatidylinositol signaling (Machida et al. We previously pointed out how ca... This idea was recently tested for farnesol’s growth inhibition of Candida parapsilosis.. protein kinase C activation (Voziyan et al.7 always safer to run a time course with and without farnesol until 90-100% germ tube formation has been achieved in the control lacking farnesol (our unpublished data). Thus. OAG and Triton X-100 showed equivalent reversal of farnesol’s growth inhibition (T. 2007). A third potential artifact is derived from farnesol’s lipophilic nature. The farnesol concentration would be effectively reduced because of the lipid binding capacity of serum albumins. no conclusions could be drawn on farnesol’s intracellular mode of action. micelle forming detergents or lipids such as DAG and OAG could incorporate the farnesol and effectively reduce its concentration. 2007) attributed internal modes of action... Butler. 2005). A similar trap could occur when farnesol’s action in blocking germ tube formation or inhibiting growth is reversed by added diacylglycerol (DAG) or 1oleoyl–2-acetyl-sn-glycerol (OAG).. i. Rossignol and G. Machida et al (Machida et al.. Farnesol Production by Candida Species . Voziyan et al (Voziyan et al. Uppuluri et al. and in this case. However. a simple way of distinguishing between reversal by an external “micelle trap” versus a more specific cytoplasmic mechanism is to find out whether equivalent amounts of detergents such as Triton X-100 and NP40 also reverse farnesol’s action. 1999. 150x more farnesol was needed to block germ tube formation in the presence of 10% serum than in its absence (Mosel et al. 1995) and Uppuluri et al (Uppuluri et al. to this DAG/OAG reversal. This prevents a misinterpretation for lack of farnesol response rather than a faster rate of filamentation. personal communication). 1999).e.
2008) but the mechanism behind . guilliermondii. 2008). 2008). krusei. Production levels for two laboratory and four clinical isolates were tightly clustered from 0. C... C.13 mg farnesol per g dry weight of fungus (Hornby and Nickerson. kefyr. parapsilosis. and C. C.8 µM farnesol while the remaining Candida species all produced < 1 µM farnesol. 2006) of 2 to 4 µM farnesol at a yeast cell density of 108/ml. GC/MS by Hornby and Nickerson (Hornby and Nickerson.7 + 3... albicans (35 µM) is substantially higher than our best estimates (Nickerson et al. 2008) recently compared the farnesol production levels from 56 strains of eight Candida species. the addition of 10% fetal calf serum (FCS) lowered farnesol production ca... based on production of ~ 0. 2004). 2008) were ca. 2-4 µM farnesol may be compatible. 2004) whereas the seven strains of C..11 to 0. This value for C. C. For example. tropicalis. 2008) varied from 13 to 58 µM farnesol. Careful consideration must also be given to the growth medium when measuring farnesol production levels. albicans studied by Weber et al (Weber et al. 2008) might be resolved by normalizing the data on a per g dry weight basis. albicans had the highest production levels (35 + 16 µM) compared to the other Candida species tested: C. the estimates of 35 µM vs.. 109/ml. 18-fold (Weber et al. Also. If the cell densities achieved following 24 hrs growth in RPMI at 37°C (Weber et al.14 mg per g dry weight (Hornby and Nickerson. glabrata (Weber et al. then the two data sets would be in remarkably close agreement. C. compared to only 108/ml in GPP at 30°C (Hornby and Nickerson. 2004). The closely related species C. dubliniensis.8 Weber et al (Weber et al. despite the different analytical procedures employed. dubliniensis produced 8. This wide variance (Weber et al. C. 2004) and derivatization with 9-anthroyl nitrile followed by HPLC by Weber et al (Weber et al.
2005). terbinafine.9 this reduction remains unclear. C. albicans thus far that regulate farnesol production: DPP3. Hornby and Nickerson. cerevisiae phosphatase Dpp1 (Toke et al. 2008. 2009) commonly used an external lipid sink such as 5% soybean oil to shift the equilibrium toward production and excretion of farnesol. 2004). Deletion of the C. tup1Δ/tup1Δ and nrg1Δ/nrg1Δ mutant strains produce 17 and 19fold higher farnesol. respectively. 2007a). Similarly. 10% FCS should have increased farnesol production rather than decreased it (Weber et al. and NRG1 (Kebaara et al. The nonspecific lipid binding capability of serum albumin was evident in the fact that 150-fold more farnesol was needed to block filamentation than in its absence (Mosel et al. albicans and other fungi with inhibitors such as fluconazole. industrial stratagems to maximize farnesol production (Muramatsu et al. Tup1 and Nrg1 appear to play a negative role in farnesol production. Possibly the low levels of farnesol detected with 10% FCS used an extraction protocol which did not fully denature the lipid-binding albumins. 2007a). 2003. Thus. than their parents.. Further . or zaragozic acid can result in increased farnesol production levels (Hornby et al. and three genes have been identified in C.. Manipulating the sterol biosynthetic pathway in C. 2008).. albicans Dpp3 is an ortholog of the S. Navarathna et al.. and it presumably acts as a phosphatase to convert farnesyl pyrophosphate to farnesol (Navarathna et al. albicans DPP3 gene results in farnesol production levels that are six times lower than wild type and parental levels (Navarathna et al. but it is unclear whether this is a result of direct or indirect sterol biosynthesis regulation (Kebaara et al. 1998). 2008).. 2007a). 2008. TUP1.... Conversely.. SQAD. The importance of a nearby lipid sink will be revisited in our discussion on the role of farnesol in pathogenicity..
. Filament and biofilm development are each blocked by farnesol in the closely related C. Farnesol and Interspecies Communication A developing branch of research has focused on the effects of farnesol signaling on interspecies communication.. Rossignol et al.. parapsilosis biofilm formation independently from filament inhibition (Laffey and Butler. many of which are above the ca. farnesol can be inhibitory or toxic. In contrast. A brief summary of the effects of farnesol on other organisms is described in Table 1.10 research is required to fully understand the regulation of farnesol production in both C. However. The use of physiologically relevant farnesol concentrations to avoid unintended cytotoxic effects will determine whether farnesol blocks dimorphism and/or biofilm formation in other Candida species. a QSM blocking . Given that the production of extracellular signaling molecules is certainly not unique to C.g.. dubliniensis. albicans and other Candida species. It is important to distinguish between general growth inhibition (an antifungal antibiotic) and specific inhibition of one type of growth. albicans morphological response to farnesol (Henriques et al. C. increasing the likelihood that the responses observed are both specific and non-specific. often accompanied by elevated reactive oxygen species (ROS) production. or detergent-like. 2005. these reports encompass a huge range of farnesol concentrations. 2007). Jabra-Rizk et al. farnesol reduces C. albicans. appears to respond in a different manner. Martins et al. e. 2007. similar to the C. 2006b. In other cell types. which normally produces very low levels of farnesol. 1 mM solubility limit for farnesol. there has also been some recent work describing the converse situation in which the response of C. albicans to bacterial signaling molecules is examined (Table 2). parapsilosis. 2007).
11 the yeast to filament transition in C. 2004). Molecules that are not inhibitory for the general growth of C. one must also consider the location of the interaction within the host. the literature has conflicting reports on the appropriate levels of farnesol with which to treat C. that same concentration also strongly inhibited growth (Boon et al. For instance (Table 2). albicans. farnesol must be used carefully and at the appropriate physiologically relevant concentrations in order to avoid artifactual observations. Similarly. even though the two molecules differ by only one methyl group. Several groups have used 150-250 µM farnesol with no apparent cell death (Davis-Hanna . Wang et al. 2008).. can inhibit filamentation without any growth inhibition (Boon et al. yet induce a signaling response are legitimate candidates for cross-kingdom signaling molecules (Table 2). albicans results in different host responses depending on the site of infection.. albicans With regard to the possibility of farnesol-mediated cell death. Lastly. An interesting consideration to make when evaluating the variety of responses to QSMs such as farnesol in different organisms is the likelihood of the cells to interact with one another during the commensal state versus during an infection. it is likely important for C. diffusible signal factor (DSF). albicans. It is intriguing how a similar molecule produced by Xanthomonas campestris. albicans commensal life style in the gastrointestinal tract (Kumamoto and Vinces.. Farnesol-Mediated Cell Death in C. 2005b) that farnesol production is turned off during anaerobic growth (Dumitru et al. as farnesol production by C. For instance.. albicans. 2008. while Burkholderia diffusible signal factor (BDSF) at a 5 µM concentration inhibited 70% of germ tube formation for C. albicans cells. such as unintended cytotoxic effects. 2004).
a fundamental question is how C. Thus. 2006b) concluded the minimum inhibitory concentration (MIC) for C. 2004).... 2008) who noted that higher concentrations of farnesol were required to block filamentation in plastic microtiter plates compared to borosilicate glass tubes or flasks.. 2009). This question is exactly analogous to an antibiotic producing strain being resistant to that antibiotic. A corollary to this idea is that reports of farnesol mediated cell death be .. with effective concentration reports ranging from 4 µM (Mosel et al. 2007.12 et al. albicans. albicans was >250 µM while another study concluded that 40 µM farnesol induced cell death (Shirtliff et al. albicans has evolved to withstand the farnesol which it produces in great abundance. Kruppa et al. Farnesol is clearly a bioactive molecule. 2008. Our summary view is that the appropriate level of farnesol to use for filament inhibition is ≤ 50 µM farnesol when using glassware. 2005). up to 250 µM (Kruppa et al. Henriques et al. albicans cultures (Weber et al. 2004). With the caveat that inappropriately high concentrations were often used. one study by Jabra-Rizk et al (Jabra-Rizk et al. suggesting a possible farnesol adsorption effect by plastic that might lower the effective concentration of farnesol. Conflicts have also emerged regarding the concentration of farnesol needed to block filamentation in C. Another important finding was that of Davis-Hanna et al (DavisHanna et al. 2004.. While the plastic versus glassware variable may explain some discrepancies in the literature.. the recurrent theme in Table 1 is that farnesol is inhibitory or lethal to a great many other cell types. albicans. this corresponds to physiologically relevant concentrations of farnesol produced by stationary-phase C. additional variables need to be considered when evaluating farnesol-mediated cell death in C. 2008).... Hogan et al.
albicans log-phase cells were considerably more sensitive to growth inhibition by farnesol than were stationary-phase cells. i. albicans. Second. in a saturated aerobically grown culture of predominantly white cells.. necrosis (Dumitru et al. Fourth. while log-phase cells grew considerably slower with 40 µM farnesol and viability was only 18% with 100 µM farnesol added (Uppuluri et al. Work is underway in our laboratories to investigate farnesol tolerance by C. The farnesol-induced death and apoptosis reported by Shirtliff et al (Shirtliff et al. albicans has let down its guard. Following this line of thinking. albicans are resistant. 2007) observed that the opaque cells were very sensitive to farnesol. 2009) in C. 2009) was for cells which had been stored for 24h in PBS buffer with no exogenous energy sources. Third. 2007).. the farnesol levels could possibly reach levels high enough to kill opaque cells.. as was first pointed out by Uppuluri et al (Uppuluri et al. Stationary-phase inocula maintained similar growth rates and viability with 0-100 µM farnesol.e. the molecular defense mechanisms are regulable rather than constant or intrinsic. ROS are generated during farnesol . At 25°C. the starting growth phase of the inoculum is critical.. in an effort to unify and validate existing data in the literature (Langford et al.13 viewed as circumstances where C. Farnesol has been reported to cause growth defects (Uppuluri et al. data not shown). 2007). only white cells of C. C. 2007) and apoptosis (Shirtliff et al. being lysed rapidly by ≥ 40 µM. First. because different conditions were used to grow the cells and different methods were used to assess cell damage.. Our conclusions so far are that four factors are significant... albicans. farnesol resistance is somewhat energy dependent. 2007). taking into account many environmental variables. Dumitru et al (Dumitru et al. although the precise concentrations required are somewhat unclear.
14 treatment of both white and opaque cells, and evidence supports mitochondrial perturbation when cytotoxic levels of farnesol are present (Dumitru et al., 2007; Shirtliff et al., 2009; Uppuluri et al., 2007). The importance of ROS is also implicit in the findings of Dumitru et al (Dumitru et al., 2007); under anaerobic conditions C. albicans tolerated mM levels of farnesol. Careful use of physiologically relevant farnesol
concentrations combined with clear indications of the previously mentioned variables will hopefully prevent future confusion on this issue. Is farnesol-mediated death in C. albicans induced in a specific or non-specific manner? Evidence exists supporting both models. Ras1/cyclic AMP signaling again becomes relevant as it is known that an activated Ras-cAMP-PKA pathway and cAMPstimulatory drugs promoted apoptotic cell death in C. albicans (Phillips et al., 2006). However, the connection of farnesol to apoptosis (Shirtliff et al., 2009) is less clear. Based on the findings of Phillips et al (Phillips et al., 2006), one would expect farnesol to lessen apoptosis rather than to promote it. Additionally, the lysis of opaque cells by farnesol was not apoptotic (Dumitru et al., 2007). Using the same procedures as had been used for showing apoptosis in A. nidulans (Semighini et al., 2006), including the definitive Annexin V test (Semighini et al., 2006), we were unable to demonstrate apoptosis in farnesol lysed opaque cells (Dumitru and Nickerson, unpublished data). Since farnesol is lipophilic, it has the ability to disrupt cell membranes, and this supports the nonspecific-mediated killing model. Understanding how farnesol regulates C.
albicans growth morphologies, growth rates, and even cell death, may be essential components for understanding the role of farnesol in the host. The Role of Farnesol In Vivo
15 Our final topic is the role of farnesol during infection. At the time of its discovery as a QSM, two ideas were put forward regarding its in vivo importance (Hornby et al., 2001). One was that, due to its ability to block the yeast to filament transition, farnesol treatment would act as a therapeutic in a mouse model of infection. The other was that farnesol would act as a virulence factor. Research on the topic has shown that the role of farnesol in vivo is not so simplistic. Navarathna et al (Navarathna et al., 2005;
Navarathna et al., 2007a) showed that farnesol acts as a virulence factor in the mouse tail vein injection assay, leading to the question of just how much farnesol is produced by C. albicans in the mouse. A definitive answer to this question will be difficult. The in vitro production estimates were for cells grown in glass flasks whereas in vivo production would be for cells growing in close proximity to mouse membranes of many types which could easily act as external lipid sinks, thus enhancing farnesol production. C. albicans is capable of evading part of the host immune system by escaping from macrophages through the production of filaments (Ghosh et al., 2009; Lorenz et al., 2004). While external farnesol reduces the viability of macrophages in vitro (Abe et al., 2009), the interesting question remains whether farnesol levels naturally produced by C. albicans are high enough to affect macrophage function/viability and thus enable fungal survival and escape from the macrophage. Does farnesol production in the phagolysosome contribute to escape from the macrophage? Experiments which add exogenous farnesol to the macrophage cannot answer this question. Also, an unfortunate obstacle for answering this question directly with the dpp3Δ/dpp3Δ mutant currently available is twofold. First, it is knocked out in only one of two genes which convert farnesyl pyrophosphate to farnesol and thus it still produces 15% as much farnesol as its
16 parent and, second, it is an arginine auxotroph (Navarathna et al., 2007a). Ghosh et al (Ghosh et al., 2009) demonstrated the requirement of arginine biosynthesis by C. albicans to effectively penetrate and escape murine macrophages. Briefly (Fig 1), Ghosh et al (Ghosh et al., 2009) showed that the rapid upregulation of arginine biosynthetic genes following macrophage ingestion, originally noted by Lorenz et al (Lorenz et al., 2004), occurred so that the arginine could be degraded to urea and then to CO2, a known signal for hyphal switching (Bahn and Muhlschlegel, 2006) necessary for C. albicans to escape. Therefore, the dpp3Δ/dpp3Δ mutant and its parental strain SN152 are unable to escape from macrophages, regardless of the presence of DPP3. New prototrophic
mutants defective in farnesol production must be constructed before this question can be answered. Consistent with early hypotheses, Hisajima et al (Hisajima et al., 2008) recently published the finding that farnesol has a protective role against C. albicans in an oral model of murine infection. This presents an intriguing conundrum: how does farnesol enhance infection for disseminated candidiasis (Navarathna et al., 2007a) while treating infection for mucosal (oral) candidiasis? A similar conundrum is how farnesol can act externally to protect yeast cells from oxidative stress (Westwater et al., 2005) but also inhibit growth by enhancing ROS production by mitochondria (Machida et al., 1998b). This suggests other factors are at play other than or in addition to the effect of farnesol on C. albicans, potentially the host’s response to farnesol. Acknowledgements We wish to thank Geraldine Butler, Tristan Rossignol, Suman Ghosh, and Raluca Dumitru for allowing us to cite their unpublished data, as well as Deborah Hogan for
A. and the Farnesol and Candida albicans Research Fund.L.L.17 helpful reading of the manuscript.).).N.). University of Nebraska Foundation (to K. . These authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.W. the Hammond-Maude Fling Fellowship (to M.L. This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.
18 Figure legends Figure 1-1. . Farnesol impacts the cyclic AMP pathway in C. albicans. Three possible points of inhibition by farnesol.
Farnesol C.2 cAMP L-ornithine Urea Tpk1/2 Car1 Efg1 L-arginine Hyphal-Specific Genes . Ras1 NH3 HCO3Cyr1 B.19 Figure 1-1 Farnesol A. Dur1.
amino acid response. parapsilosis Acinetobacter baumannii Aspergillus fumigatus Aspergillus nidulans Farnesol Concentration(s) Summary of Farnesol Response Used 1. altered spore germination and lysis µM (Semighini et al. apoptosis (Semighini et al. 2006). 2008) Inhibition of conidiation (Lorek et al.10mM 100 µM . 2009) Inhibition of Pseudomonas quinolone signal (PQS) and pyocyanin production.250 µM 1 µM . hyphal inhibition (1-10 µM) (Semighini et al. 2005).. 2007..300 Apoptosis. 2008) Aspergillus niger Fusarium graminearum Human gingival cells Human oral carcinoma cells Murine macrophages Paracoccidiodes brasiliensis Pseudomonas aeruginosa Saccharomyces cerevisiae 100 µM.... growth arrest ≥50 µM. growth inhibition at high concentrations (Derengowski et al. Organism C.300 µM Reduced proliferation and adhesion (Saidi et al. dubliniensis C. no effect on growth.300 µM Apoptosis. lipid metabolism. 2008) 10 µM . ribosome biogenesis. 2007) 50 µM. 1999).5 mM .. 2007. 2006) 10 µM . Martins et al..112 µM 5 µM . effect on mitochondria and increased reactive oxygen species (ROS) production (Machida et al.5 nM.... 2006). decreased swarming motility.. and translation genes affected (10-100 µM) (Savoldi et al... cell death (Fairn et al.. The effects of farnesol treatment in other cell types. McAlester et al. 2008) 56 µM . 2006) Apoptosis and ROS production (10 – 250 µM) (Semighini et al. autophagy. 2006b) 1. 5 µM. (Cugini et al.200 µM Growth inhibition (Peleg et al.300 µM Filament inhibition. 2009) Inhibition of yeast to hyphal and hyphal to yeast growth at low concentrations. and amino acid biosynthesis genes affected. biofilm inhibition.250 µM Growth inhibition. 2008) 10 µM.. lipid metabolism.. 2007).5 nM-100 µM Biofilm inhibition (Laffey and Butler. 1998a. (Henriques et al. 2008) Growth inhibition by cell cycle arrest. Machida et al. ROS production.. decreased phagocytic activity (Abe et al...300 µM 30 µM . MIC = 200µM.. altered lipid polarization (Rossignol et al. transcription.60 µM Apoptosis (Scheper et al. lowered tolerance of fluconazole (Jabra-Rizk et al.20 Table 1-1.
2006) Staphylococcus aureus Staphylococcus epidermidis Streptococcus mutans Tobacco (Nicotiana tabacum L.. 2007. 2003.. biofilm inhibition. 2008) Cell death (Gomes et al. cv Bright Yellow2) 30 µM . 2002a. biofilm inhibition..21 2007) Cell death. 2009) Growth inhibition. increased sensitivity to antibiotics (Jabra-Rizk et al.. 2002b. Koo et al. Kuroda et al. membrane disruption. prevention of dental caries in rats when in combination with fluoride and apigenin (Koo et al.5 mM 5 µM -150 µM .4..5 mM 100 µM -200 µM 250 µM . Koo et al. 2005) Cell death (Hemmerlin and Bach. 2006a. lipase inhibition. Hemmerlin et al.. Koo et al.. Togashi et al.. 2000..
. BDSF) 3-oxo-C12homoserine lactone. biofilm inhibition.. 2004) . and reduced viability (Tampakakis et al. 2009) Induction of filamentous growth. found in cell-free supernatant Unknown.. albicans Response Filament inhibition. found in cell-free supernatant and missing/reduced in ΔluxS mutant Cis-11-methyl-2dodecenoic acid (diffusible signal factor. found in cell-free supernatant Cis-2-dodecenoic acid (Burkholderia diffusible signal factor. The effects of secreted bacterial molecules on C. albicans. 2008. DSF) Summary of C. Hogan et al. Wang et al. aeruginosa Salmonella enterica serovar Typhimurium Streptococcus gordonii Xanthomonas campestris Secreted Molecule(s) Unknown.22 Table 1-2. 2009) Filament inhibition (Boon et al... Bacterium Acinetobacter baumannii Burkholderia cenocepacia P... 2004) Cell death (Gibson et al. 2008) Filament inhibition and reversion to yeast morphology (Davis-Hanna et al. biofilm inhibition (Peleg et al. and C12acyl homoserine lactone Phenazines Unknown. aeruginosa P. 2008. 2009) Filament inhibition. dodecanol. 2008) Growth inhibition and filament inhibition (Boon et al. suppression of farnesol-mediated filament inhibition (Bamford et al...
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K.L. 54(2): 940-2. Hasim. A.W. and Atkin..L. .35 CHAPTER 2 Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions Reference: Langford. Nickerson.. Antimicrob. S. M. Agents Chemother. (2010) Activity and toxicity of farnesol on Candida albicans is dependent on growth conditions..
This study provides a clear understanding of what farnesol concentrations are lethal to C. and media were altered. validating previous studies and identifying new variables. Parameters for farnesol tolerance/sensitivity are defined. temperature. albicans. Farnesol sensitivity was measured when inoculum cell history and size. but confusion abounds regarding which conditions promote these distinct responses. . based on environmental conditions.36 Abstract Farnesol interacts with Candida albicans as both a quorum sensing molecule and toxic agent. such as energy sources.
. Aspergillus nidulans (Semighini et al. One feature of farnesol’s action as a QSM was that exogenous farnesol up to 200-300 μM does not alter the growth rate. 2005).. 2009). and Kruppa et al (Kruppa et al. These results were quickly confirmed by Ramage et al (Ramage et al.. Farnesol production is regulated in that it is turned off in opaque cells (Dumitru et al. Paracoccidioides brasiliensis (Derengowski et al. 2001). instead. C. It is also the model system for studying quorum sensing for fungal dimorphism (Hornby et al. albicans excretes a quorum sensing molecule (QSM) called farnesol and when extracellular levels exceed a threshold of 1-5 μM (Mosel et al... and is a protective factor during mucosal infection (Hisajima et al. albicans biology: it blocks biofilm formation (Ramage et al. 2007) and during anaerobic growth (Dumitru et al. 1999). 2004). 2007a). Navarathna et al... 2005).. Farnesol is a bioactive molecule with mild detergent-like properties..37 Introduction Candida albicans is a dimorphic fungus of great medical importance.. 2002). Machida et al. 2008).. 2008). Hogan et al (Hogan et al.. 2004. 2004). 2002). Farnesol also impacts other aspects of C... At concentrations in the 20-50 μM range it has been reported to inhibit or induce cell death in Saccharomyces cerevisiae (Machida et al. 1998a. Farnesol production also increases in the presence of sublethal levels of sterol biosynthesis inhibitors (Hornby and Nickerson. 2004) but elevated in some mutants which are altered in morphology (Jensen et al. and C. farnesol blocks the yeast-to-filament conversion. 2008). the cells grow as yeasts rather than as filaments. Fusarium graminearum (Semighini et al... acts as a virulence factor during systemic infection (Navarathna et al... albicans opaque cells . 2006) or locked in the filamentous morphology (Kebaara et al. 2006).
2009) who reported that farnesol. This view was recently challenged by Shirtliff et al (Shirtliff et al. 2006a.38 (Dumitru et al. albicans exhibited exceptional tolerance to farnesol.. washed and resuspended in PBS for farnesol sensitivity assays. innocuous to growth. Similarly. 2008). . Jabra-Rizk et al. 2006b.. albicans by inducing apoptosis. 2009. farnesol has been reported to trigger cell death in several mammalian cell lines (Abe et al.. 2008) and bacteria (Gomes et al. Koo et al.. 2007.. and the conditions under which Shirtliff et al (Shirtliff et al. 2009. adding to confusion on the matter of farnesol-induced cell death. Previous studies examining C. killed C. 2009) observed cell death.. the view to this point was that C. Shirtliff et al. 1993. 2007) have used vastly different assay conditions (varying temperatures.. we were particularly interested whether farnesol resistance in C... Thus. at concentrations as low as 40 μM.. by macrophages. 2003).. 2009) used cells that were grown overnight. Uppuluri et al. in the spirit of constructive dialogue.. 2007). Rajagopal et al. albicans sensitivity to farnesol (Dumitru et al. Togashi et al. and it induces cytokine production. 2009. Saidi et al. Thus. albicans is similarly energy dependent. and inoculum growth phases). such as IL-6. Critically. Since it is well known that detergent resistance in bacteria is an energy dependent process (Aspedon and Nickerson.. Jabra-Rizk et al. media.. 2003.. we draw attention to the differences between the growth conditions used in our previous body of work under which farnesol would act as a signaling molecule. 2006.. Shirtliff et al (Shirtliff et al. inoculum sizes. Scheper et al. as a necessary corollary to its production as an antagonistic molecule.
E-farnesol in methanol so that the final methanol concentration never exceeded 1%.5).. growth medium (rich vs. cell density. 2009). cultures were inoculated to an OD600 =0.39 Methods We examined farnesol sensitivity under a series of different growth conditions in plastic microtiter dishes. and inoculated at the indicated levels with variable concentrations of farnesol. . minimal). albicans cells were grown to stationary phase (unbudding cells. We followed cell growth by means of optical density and cell death by methylene blue staining (Gibson et al. and inoculum history. C. Variables included temperature.1 and grown at 30°C for 16-18h) or mid-log phase (OD600 =0. washed three times in PBS. and this level of methanol had no effect on cell growth or cell death (data not shown). We used 10 mM and 100 mM stocks of E.
40 Results In rich growth medium (YPD), no cell death was observed at farnesol concentrations up to 300 μM and growth inhibition was only observed with 300 μM farnesol (data not shown). When we switched to a defined glucose-proline (GPP pH6.8) (Kebaara et al., 2008)medium (Fig. 1), very similar growth curves were observed with all concentrations of farnesol up to 300 μM when starting with stationary phase inocula (Fig. 1A). The cell growth experiments (Fig. 1) were simultaneously examined for cell death by staining with methylene blue (Fig. 2B, 2D). Minimal cell death occurred in GPP with a stationary phase inoculum, our standard growth conditions (Hornby et al., 2001; Mosel et al., 2005), and up to 300 μM farnesol (Fig. 2D) consistent with growth curves seen in Fig. 1A. However, when we used inocula of exponentially growing cells, 40 μM farnesol partially inhibited growth and further inhibition correlated with increasing farnesol levels (Fig. 1B). Log phase cells were killed by 100 and 300 μM farnesol in GPP (Fig. 2B), consistent with the delayed growth seen in Fig. 1B. These results support the growth phase-dependent sensitivity described by Uppuluri et al (Uppuluri et al., 2007). Temperature does not play a prominent role in farnesol growth inhibition because we obtained similar growth curves at 25ºC, 30°C, and 37°C (data not shown). To examine the effects of different media on farnesol sensitivity, cells were compared under both growth (GPP) and storage (PBS) conditions, using both exponential and stationary phase inocula (Fig. 2). For exponential phase cells inoculated in PBS, even low levels of farnesol, i.e. 40 μM, caused cell death (Fig. 2A), consistent with the findings of Shirtliff et al (Shirtliff et al., 2009). The cells in PBS were far more sensitive to farnesol when they had come from an exponential phase inoculum than a stationary
41 phase inoculum (Fig. 2A, 2C). Interestingly, both exponential and stationary phase cells showed increased tolerance to farnesol when incubated in growth media (GPP or YPD), compared to PBS (Fig. 2). As with growth rates, similar results for cell death were obtained at all 3 temperatures tested, 25°C, 30°C, and 37°C. These observations suggest a role for energy source(s) in C. albicans farnesol tolerance. The previous experiments (Figs. 1 and 2) were conducted in 96 well plates with farnesol added at time zero to washed cells. Because of a possible farnesol adsorption effect with plastic (Davis-Hanna et al., 2008), we confirmed the farnesol sensitivity of exponentially growing cells in glass flasks (Fig. 3). We compared the farnesol sensitivity of C. albicans cells in exponential phase (OD600 = 0.5, Fig. 3A, 3B) and stationary phase (OD600 = 4.0, Fig. 3C, 3D) by adding farnesol directly to unwashed growing cultures. The results with glass flasks (Fig. 3) were consistent with those obtained with plastic 96 well plates.
42 Discussion A distinct benefit of this controversy is to focus attention on the conditions in which C. albicans tolerates farnesol while so many other cell types are killed by it. Stationary phase C. albicans cells exhibit extreme farnesol tolerance, even at 100-300 μM farnesol, and these levels are lethal to most other organisms tested. Throughout, cell death was not accompanied by cell lysis because there was no drop in OD600 and the methylene blue positive cells remained intact. Similarly, optical densities of cells in PBS were not significantly affected by the presence of farnesol (data not shown). This lack of cell lysis with white cells of C. albicans is in marked contrast with opaque cells where ≥ 40 µM farnesol caused rapid cell lysis (Dumitru et al., 2007). The differences in
susceptibility between white and opaque cells (Dumitru et al., 2007), aerobic and anaerobic cells (Dumitru et al., 2004), stationary and exponential cells [(Uppuluri et al., 2007) and Figs. 1-3], and between cells in buffer alone versus minimal media suggest a physiological adaptation to farnesol, depending on environmental conditions. Two places where this selective farnesol tolerance might be localized are the mitochondria, where farnesol stimulates ROS production in C. albicans and S. cerevisiae (Machida et al., 1998a; Machida et al., 1999; Shirtliff et al., 2009), and the lipid portion of the cytoplasmic membrane. Changes in fatty acid composition, sterol-rich domains (Alvarez et al., 2007), sphingolipids (Brown and London, 2000), and GPI-anchored proteins preferentially associated with lipid rafts (Brown and London, 2000) could be involved, as supported by the lipid domain alteration seen in C. parapsilosis after farnesol treatment (Rossignol et al., 2007). Although the mechanisms(s) involved in farnesol tolerance
30°C.L. Understanding these environmental parameters may unify many previous discrepancies in the literature and provide unambiguous conditions to induce each of farnesol’s unique effects on C.). albicans.A.10. the Hammond-Maude Fling Fellowship (to M. under energy-starved conditions. University of Nebraska Foundation (to K. optimal conditions to examine farnesol-mediated cell death include the use of log phase cells in PBS. as these do not undergo cell death with high farnesol concentrations. stationary phase cells inoculated into minimal or rich media are ideal candidates for studying farnesol signaling. Conversely.N. .L. conditions in which C. albicans is either sensitive or resistant to farnesol have now been outlined. 37°C) as well as for experiments with starting cell densities of OD600 = 0.).43 remain unclear.05 or 0. and the Farnesol and Candida albicans Research Fund. In summary.L.W. This generalization was true for all three commonly used temperatures (25°C. Acknowledgements This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.).
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B) or stationary phase (C. Incubations were initiated with either exponential (A. 2-2. D) was subdivided when it had reached stationary phase (OD600 = 4. A) stationary phase inoculum.0). 2-1. and OD600 values were recorded on an automated plate reader (Molecular Devices. Sunnyvale. Fig. . 2009). C) and % dead cells (B. Cells were incubated in either PBS (A. Toxicity of farnesol to exponential cultures of C. 2-3. D) inocula.49 Figure Legends Fig. B) was subdivided (in six) when it had reached exponential phase (OD600 = 0. Percent death was determined by methylene blue staining (Gibson et al. Note the different y-axis scales in graphs A and B. Cells were grown in duplicate on at least two separate occasions in defined GPP medium with indicated levels of farnesol (see legend in B) at 30°C in 96-well plates. All cultures were in glass flasks in GPP both before and after subdivision. albicans cell growth. Effect of farnesol on C. Cultures containing the indicated levels of farnesol were shaken at 30°C and 250 rpm for 4 hrs and at the indicated times cell growth (A. CA). D) were determined.5) and the other (C.. Effect of farnesol on C. C) or GPP (B. One culture (A. albicans. Cultures were not washed prior to subdivision and farnesol addition. albicans cell death. D) with the indicated levels of farnesol in 96-well plates at 30°C. Fig. B) exponential phase inoculum.
50 Figure 2-1 .
51 Figure 2-2 .
52 Figure 2-3 .
Langford.. M. Nickerson. D.W.53 CHAPTER 3 Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentousgrowth induction Reference: Kebaara. Cell.L. . K. Dumitru.. R... B.L. Navarathna.H.W. and Atkin. A. (2008) Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction. 7: 980-7.. Eukaryot.
Tup1 functions with the DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress expression of hyphal specific genes. NRG1. Its ability to regulate morphogenesis is strongly correlated with virulence. than the parental strain. Based on this overlap in function. albicans. HWP1 and RBT1. Farnesol did not affect EFG1 (a transcription regulator of filament development). albicans cells with farnesol caused a small but consistent increase in both TUP1 mRNA and protein levels. we tested the hypothesis that the cellular response to farnesol involves. the tup1/tup1 and nrg1/nrg1 mutants produced 17. Our data are consistent with Tup1 being a crucial component of the response to farnesol in C. Treatment of C. The tup1/tup1 and nrg1/nrg1 mutants.54 Abstract Candida albicans is a dimorphic fungus that can interconvert between yeast and filamentous forms. These levels of excess farnesol are sufficient to block filamentation in a wild-type strain. Tup1.and 19-fold more farnesol. this increase corresponds with the commitment point. the activation of Tup1. a transcriptional repressor. beyond which added farnesol no longer blocks germ tube formation. in part. Tup1 probably plays a direct role in the response to farnesol because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. . Importantly. hyphal-specific genes. or RFG1 mRNA levels. Further. demonstrating specific gene regulation in response to farnesol. respectively. and it correlates with a strong decrease in the expression of two Tup1regulated. failed to respond to farnesol. but not the rfg1/rfg1 mutant. and the signaling molecule farnesol are both capable of negatively regulating the yeast to filamentous conversion.
2001).. E) farnesol (Hornby et al. and the mortality rates for patients with candidiasis are 30-50% even with antifungal treatment (Kullberg and Filler.. 2005.. indicating a need for new antifungal drugs. 1992).. from mild mucosal infections to life-threatening systemic infections termed candidemia (Jarvis and Martone. 2003) and. 2001). Stationary phase cultures of C. However. C. as well as on the skin. and when it accumulates beyond a threshold level it blocks the yeast to filament conversion (Hornby et al. 2001). 2002). Mosel et al. 1992). albicans is capable of causing a wide range of disease. these farnesol production levels are physiologically relevant. The ability of C. 2002). albicans is part of the normal flora.55 Introduction Candida albicans is the most commonly isolated opportunistic fungal pathogen in humans. Other roles described for farnesol include biofilm inhibition (Ramage et al. our research has focused on farnesol.... and organ transplant patients (Jarvis and Martone. patients undergoing chemotherapy. 2007a) that is excreted continuously by C. 2001) and the IC50 value for blocking germ tube formation (GTF) in an N-acetylglucosamine stimulated assay is ca. albicans have accumulated 2-4 µM farnesol (Hornby et al. Farnesol is a virulence factor (Navarathna et al. Vulnerable patients include AIDS patients. 2001.. 1-2 µM (E. albicans (Hornby et al. the first quorum sensing molecule (QSM) discovered in a eukaryote (Hornby et al.. protection from . consequently. Recently. and it resides in the gastrointestinal and genitourinary tracts. The annual cost of treating candidiasis in the United States was estimated to be one billion dollars. Shchepin et al. albicans to cause disease has been strongly linked to its conversion between two distinct morphological forms: yeast and filaments. C.
2002... Aspergillus nidulans (Semighini et al.. Calcium/Calmodulin signaling pathway. Farnesol is able to block filamentous growth induced by environmental signals for most. In addition to farnesol. Mechanisms have also been identified that block filament development with transcriptional repression by Tup1 playing a key role (Braun and Johnson.. Westwater et al. and Chk1 twocomponent signal transduction pathway. 2006). 2007). Kumamoto and Vinces.. 2004. Kruppa et al. 2000). The yeast to filamentous conversion is activated by many pathways. While many phenotypic effects produced by farnesol have been described. 1998. Efg1 also regulates the expression of multiple genes including those involved in virulence (Eckert et al. and possibly all of the signaling pathways activating filament development. Rim101-independent pathway. Activation and inhibition of filament development is largely accomplished through changes in gene expression mediated by transcription activators and repressors. Dhillon et al.5 mM N-acetylglucosamine.. 2007. 2. 2005a). 1997. 2010. These signals include: 10% serum. 10 mM L-proline. 2003). Ras/cAMP-dependent pathway. Efg1 is a major transcription regulator of filamentous growth and is a central control point for many signaling pathways involved in filamentation (Eckert et al.56 oxidative stress (Deveau et al. Braun and Johnson. and induction of apoptosis in another fungus. 2005). these pathways show some degree of specialization in that they respond to different environmental inducers.. Although each has been implicated in filamentation (Csank et al. including: Components of the CEK1 MAP kinase pathway. C. albicans yeast and filamentous growth is controlled by an assortment of signaling pathways (Berman and Sudbery. 1999).. little is understood about farnesol’s mode of action. Ramon et al.. or the .
and rfg1/rfg1 strains to assess the requirement for these genes in the farnesol response. 2001b). albicans response to farnesol involves Tup1. Sprague et al.. albicans Tup1 protein is a transcription regulator that plays two key roles in the cell: 1) regulation of phase switching. Rfg1 (homologous to S. cerevisiae Mig1p). Murad et al. The C. 2006. all at 37oC (Hornby et al. 2001a. Braun and Johnson. 2000).. and 2) inhibition of filamentous growth. Tup1 repression of filament specific genes is an attractive candidate for a common control point that may be regulated by farnesol (Kadosh and Johnson. Tup1 interacts with either Ssn6 or Tcc1 corepressor proteins. Deletion of TUP1 results in the up- regulation of approximately one-third of C. cerevisiae Rox1p). 2001b) and these mutants are also avirulent in a murine model of infection. we tested the hypothesis that the C. At least three DNA-binding proteins have been identified that function with Tup1: Nrg1 (homologous to Saccharomyces cerevisiae Nrg1p).. Thus. tup1/TUP1.. This complex functions with DNA binding proteins to repress gene expression (Kaneko et al. The gene expression pattern for MIG1 was not determined because . 1997). 2001a. Murad et al.57 combination of N-acetylglucosamine and L-proline. 2005). farnesol may individually block each of the morphogenic signaling pathways and/or act at a common control point in morphogenesis. Murad et al. The morphological response to farnesol was tested in wild-type.. and Mig1 (homologous to S. albicans genes (Murad et al.. 2000. tup1/tup1.. 1997. Activation of Tup1 transcription repressor complexes results in the repression of filament-specific gene expression (Braun and Johnson. Homozygous tup1 mutants are unable to grow as yeast and instead remain locked in the filamentous form in all media tested (Braun and Johnson. Here. nrg1/nrg1. 2001).
. we compared farnesol production levels in tup1. RFG1. 2001a). as well as genes under their control were examined in the presence or absence of farnesol by quantitative northern and western analyses. albicans (Murad et al. and EFG1. . nrg1. Finally.58 Mig1 protein does not play a role in filamentous growth of C. and rfg1 homozygous mutants relative to wild type cells. The gene expression patterns of TUP1. NRG1.
MnCl2. in CAI-4.5 mM N-acetylglucosamine (Hornby et al. 2001).59 Methods Strains and media Candida albicans SC5314 is an independent clinical isolate and the reference strain for the Candida genome sequence. 200 μg.. MAL3:p455. albicans strains CAF-2 (ura3:imm434/URA3) and CAI-4 (ura3::imm434/ura3::imm434) are derived from SC5314 by gene replacement . Dunedin. L-proline. ZnSO4•7H20. (Braun and Johnson. thiamine•HCl. in CAI-4. 1981). in CAI-4.8 contained 3. biotin. 1. Strains BCa2-9 (tup1/tup1.. CuSO4•5H20. as were the filter-sterilized vitamins (Kulkarni and Nickerson. Modified GPP (mGPP) also contained 2. DU129 (rfg1/rfg1. NaH2PO4. BCa05 that expresses TUP1 ectopically (tup1/tup1. in CAI-4. 2001).8 contained (per liter distilled water): glucose. The glucose (20% w/v) and L-proline (100 mM) were autoclaved separately and added aseptically. FeSO4.5. (Braun and Johnson. 4g. 1997). New Zealand.2g/L Na2HPO4 . 1mg. 20 μg. in CAI-4. 0. University of California. frameshift disruption fragment in CAI-4. and stored at 4°C to be used within a month. KH2PO4. BCa2-10 (tup1/tup1.2g. MgSO4•7H20. GPP pH 6. resuspended in 10 ml of 50 mM phosphate. 1mg. washing three times with 50 mM phosphate pH 6. 20g. Resting cells were obtained by growing cells in modified glucose salts biotin media (mGSB) overnight. C.5g. 1mg. DU152 (nrg1/nrg1. pyridoxine•HCl. 1 mg. Kadosh and Johnson. (Backen et al. 1997). (Braun and Johnson.15g. 1997) were obtained from Alexander Johnson. 200 μg. 1997). 3. The defined glucose-salts medium GPP pH 4. University of Otago. San Francisco. CA. (Braun and Johnson. 1997) and BCa2-3 (TUP1/tup1.. (Braun and Johnson. 2000. Strain MEN was provided by Richard Cannon.
Piscataway. The Northern blots were probed with radiolabeled DNA probes.. Boston. All media for CAI-4 included uridine at 40 μg/ml (Backen et al.. Equal amounts of RNA (15μg) were resolved on 1.5-0. NJ) and quantified using ImageQuant software (Molecular Dynamics version . Northern blots were PhosphorImaged using a Storm (Amersham Pharmacia Biotech Inc.5% (w/v) agar. Solid media included 1. Kebaara et al. Inc. Microscopy Differential interference contrast (DIC) images were photographed with an Olympus BX51 microscope and colony morphology images were photographed with an Olympus SZX12 microscope.. 2000. Cells were grown at 37°C for 0. 20. MI) was also used. 2000). 60. MA) using the capillary blot transfer protocol recommended by the manufacturer. CA). Quantitative Northern blot analysis To measure mRNA accumulations. RadPrime DNA labeling system following the protocol recommended by the manufacturer (Invitrogen. Detroit.. The probe DNAs used for synthesis were prepared by PCR using MEN genomic DNA.60 instead of NaH2PO4.6 and allowed to equilibrate at 37°C for 5 minutes whereupon 20 μM farnesol was added to half of the flasks. Carlsbad. The probes were labeled with 32 P-dCTP (GE Health Sciences. SC5314 resting cells were inoculated in mGPP to an OD600 of 0. 40. For maltose phosphate proline media (MPP). filter-sterilized maltose replaced the glucose. and 80 minutes until the cells were harvested and total RNA extracted by the hot phenol method (Backen et al. Piscataway.0 % agarose-formaldehyde gels and the RNA was transferred to GeneScreen Plus (NENTM Life Science Products. 2003). Cornmeal agar (Difco.. NJ) using an oligolabeling kit.
(Piscataway. 2002). NJ). Analysis of farnesol levels Extracellular farnesol was extracted from cell free-supernatants of cultures grown in mGPP at 30o and analyzed by GC/MS as described (Hornby et al. HRP labeled anti-mouse antibody was from Perkin-Elmer (Boston. 1995) and Tup1 and Act1 proteins detected with the Supersignal® West Pico Chemiluminescent Substrate using the manufacturers protocol (Pierce.0.. CA). Sunnyvale. IL) with the exception that blocking was done with 5% nonfat dry milk. Western blot analysis Western blots were prepared as previously described (Atkin et al. 2001). Rockford.. mRNA abundance measurements were done using a minimum of three independent Northern blots. . Rabbit polyclonal antibodies against Tup1 were previously described (Inglis and Johnson. Mouse monoclonal Anti-Act1 antibodies and horseradish peroxidase (HRP) labeled ant-rabbit IgG antibodies were from Amersham Pharmacia Biotech Inc. MA).61 5.
1997) restores the ability to respond to farnesol (data not shown). Nrg1 and Rfg1.62 Results tup1/tup1 and nrg1/nrg1 mutants lack a morphological response to farnesol while the rfg1/rfg1 mutant responds to farnesol The juxtaposition of farnesol’s ability to inhibit differentiation. albicans SC5314 in filament-inducing media grew as yeasts in the presence of 20 μM farnesol and as filaments in media lacking farnesol. As a control. and the role of Tup1 as a transcription repressor for filamentation genes suggests that farnesol could function by activating Tup1 and/or one of its co-regulators. Khalaf and Zitomer. 2001a. tup1/tup1 and nrg1/nrg1 mutants differ from the great majority of filamentous-only mutants recovered from a previous study. NRG1. The filamentous-only cell morphology is the expected phenotype for these known mutants (Braun and Johnson. the lack of response to farnesol was specific for loss of Tup1 because we found ectopic expression of TUP1 (Braun and Johnson. Murad et al. 2001. Murad et al. demonstrating a positive response to farnesol (Fig. . in this regard. Consequently. we examined the effect of farnesol on the morphology of null mutants lacking TUP1.. Braun et al. 1). 1). For the tup1/tup1 mutant. 2006). 2001b). the wild type C.. Unlike SC5314 and rfg1/rfg1. The rfg1/rfg1 mutant responded to 20 μM farnesol in a similar manner to SC5314 (Fig. and RFG1. However.. 1). 96% of which reverted to a smooth colony (yeast) morphology on YM agar plates with 50 μM farnesol (Jensen et al. 1997. the tup1/tup1 and nrg1/nrg1 mutants lacked a detectable response to farnesol and remained filamentous in the presence of 20 μM farnesol (Fig.. 2001.
5±0. 60 and 80 min following induction.. 2005). n=4) in TUP1 mRNA levels from 20 to 60 min (Fig 2A). in these conditions. the TUP1 mRNA levels decreased over the first 20 min and then increased (Fig.63 TUP1 mRNA levels increase in the presence of farnesol while RFG1 and NRG1 mRNA levels were not affected by farnesol We analyzed the effect of farnesol on TUP1 mRNA levels over time in C.3. 2005) that.. we found that TUP1 mRNA consistently increased 2. Importantly. This . 40. farnesol (20 µM) causes a consistent increase in TUP1 mRNA levels during the precise time period when it blocks differentiation from yeasts to filaments. (Toyoda et al. this is the time period just prior to that at which the cells become committed and are no longer responsive to farnesol (Mosel et al..8) at 37°C in the presence and absence of 20 μM farnesol and mRNA levels were determined at 0. 2004) who showed that TUP1 mRNA levels increased slightly at 180 minutes after induction of filamentation. 20. albicans SC5314 cells which had been induced to differentiate from yeasts to filaments by growth at 37°C in mGPP. Thus. farnesol no longer blocked germ tube formation when added 60-90 min after inoculation (Kulkarni and Nickerson. 2A).6 (n=4) fold from 20 to 60 min. We previously showed (Mosel et al. there was very little increase (1. Here our analysis was designed to evaluate changes in TUP1 mRNA just before germ tube formation when the cells were still responsive to farnesol.4±0. Further. In the presence of farnesol. This pattern is consistent with the single time point results of Toyoda et al. 1981). In all experiments. in the absence of farnesol. Filamentation was induced by transferring resting cells into mGPP (pH 4. In contrast. the first germ tubes appeared at 30 min and the process was complete by 110 min.
At 80 minutes. 2B and C). we conclude that farnesol does not affect RFG1 or NRG1 mRNA levels. albicans strain JCM9061 NRG1 mRNA levels decreased during filamentation (Toyoda et al. in farnesol treated than untreated cells. we examined the expression of two Tup1-regulated genes. (De Groot et al. and in C. Farnesol delays and dramatically reduces the magnitude of HWP1 and RBT1 mRNA expression (Fig. we tested the effect of farnesol on RFG1 and NRG1 mRNA levels during differentiation from yeasts to filaments.3).. RFG1 mRNA levels initially decreased and then increased (data not shown). Under the same conditions.5-fold in TUP1 mRNA corresponded to an increase in SC5314 Tup1 protein levels at 60 min following induction (Fig. 2004). the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig.6-fold lower. NRG1 mRNA levels did not change during development and they too were the same in the presence and absence of farnesol (Fig.. data not shown). Similar results were observed by Davis-Hanna et al. unlike TUP1. respectively. . Since Tup1 functions with DNA binding proteins such as Rfg1 or Nrg1.and 7.. HWP1 and RBT1 levels were 30.5-fold. However. 2003)]. Thus. 2B and C). 4). 2B and C. Expression of the Tup1-regulated filamentous genes HWP1 and RBT1 is inhibited by farnesol To test whether the increased TUP1 expression in the presence of farnesol was biologically significant. (Davis-Hanna et al. Tup1 protein in SC5324 was increased in all three replicate experiments by an average of 2. In the absence of farnesol. HWP1 and RBT1 [Fig. the HWP1 and RBT1 transcripts were undetectable at time 0 but they were strongly expressed from 40 to 80 minutes (Fig.64 increase of 2. Like TUP1. 4.
2003). 4). To test this hypothesis. Thus there is a strong correlation between elevated TUP1 expression in response to farnesol and the expression of Tup1-regulated genes. 1997). and then increased steadily throughout the remaining time (data not shown). 1997) showed that BCa2-3. EFG1 mRNA levels remain unaffected by farnesol Efg1 is a transcription regulator for genes required for filamentation. Farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote. Braun and Johnson (Braun and Johnson.. we . we tested whether farnesol also affects EFG1 mRNA levels (Fig. 3). Presumably these cells do not make enough Tup1 to compensate for the reduced gene copy number. farnesol had no influence on EFG1 mRNA levels since the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig. decreased to a minimum at 20 minutes.65 2008) for HWP1 mRNA 2 hours after treatment with 75 μM farnesol. a TUP1/tup1 heterozygote. We hypothesized that farnesol might suppress this phenotype because it increases TUP1 expression 2. The EFG1 mRNA levels were high at time 0.5-fold in SC5314 and 4.4 and data not shown). EFG1 mRNA levels are down regulated at the initiation of filament development and then increase as filament formation progresses (Tebarth et al. HWP1 and RBT1 are activated by Efg1 during filamentation. This increase should restore Tup1 to roughly wild type levels. is haploinsufficient in that these cells develop a higher proportion of filaments compared to wild-type cells on most media (Braun and Johnson. However.2 fold in TUP1/tup1 (Fig. Therefore.
in the presence of farnesol the TUP1/tup1 mutant looked identical to wild type C. Farnesol production levels were dramatically increased in the tup1/tup1 and nrg1/nrg1 mutants (Table 1) that were unable to respond to farnesol (Fig. In contrast to the haploinsufficient phenotype observed in the absence of farnesol. This overproduction suggests the ability to respond to farnesol may be linked to regulation of farnesol production. the farnesol responsive rfg1/rfg1 mutant only produced ca. The tup1/tup1 and nrg1/nrg1 mutants produced ca.and 19-fold more farnesol. respectively. CAF-2. and Tup1 protein levels were ca. 1). A subset of these mutants produced levels of farnesol significantly higher than wild type strains. 1). albicans (Table 2). the addition of 20 μM farnesol results in growth as yeasts (Fig. tup1/tup1 and nrg1/nrg1 mutants produce excess farnesol.2-fold higher in the TUP1/tup1 mutant treated with farnesol. 2. Jensen et al (Jensen et al. albicans BCa2-3 on cornmeal agar plus Tween 80 under a coverslip for 25 hours at 25°C. Thus.6-fold more farnesol than the wild-type strains (Table 1).. Here we tested farnesol production levels in CAI-4. 4 and Table 2). 2006) tested the farnesol production levels for several filamentous-only mutants. and rfg1/rfg1 strains. In contrast. although it forms filamentous cells when grown in mGPP. Thus farnesol suppresses the haploinsufficiency phenotype of the TUP1/tup1 heterozygote. tup1/tup1.66 examined the effect of farnesol on C. the TUP1/tup1 mutant was more filamentous than wild-type colonies but less filamentous than the tup1/tup1 mutant (BCa2-10. nrg1/nrg1. we showed the TUP1/tup1 mutant responds to farnesol because. As a control. the two mutants . In these conditions. 17. than did CAF-2 and CAI-4. 4.
67 that are unable to respond to farnesol (tup1/tup1 and nrg1/nrg1) produced much higher levels of farnesol than did strains that do respond to farnesol. tup1/tup1 overproduction of farnesol inhibits SC5314 filamentation. 5. When SC5314 was plated and followed one day later by another streak with SC5314. We tested the biological significance of farnesol overproduction by sequentially plating tup1/tup1 and SC5314 next to one another and observing the resultant colony morphologies. followed by SC5314. a much larger area of filament inhibition was observed (Fig. In contrast. . These results are consistent with the tup1/tup1 overproduction of farnesol. no filament inhibition was observed (Fig. 5A). C and D). a small area of filament inhibition was observed (Fig. when tup1/tup1 was plated first. 5B). As controls. whenever tup1/tup1 was plated second.
. by changing gene expression (Cao et al. Finally. and 3). HWP1 and RBT1 mRNA levels decrease (Fig. Importantly.. Further. 2005) and during resumption of growth following stationary phase (Enjalbert and Whiteway. Tup1 mRNA and protein levels increased in the presence of farnesol while two Tup1-regulated genes. we believe that Tup1 is part of the farnesol response pathway because farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 strain (Table 2). 2005). Previous work examining farnesol- dependent changes in the global transcription profiles of developing biofilms (Cao et al. 2005). the timing of this increase (40-60 min. Fig. Enjalbert and Whiteway. Cell synchrony.. Here we show that the tup1/tup1 and nrg1/nrg1 null mutants are strictly filamentous and the cells remain filamentous in the presence of added farnesol (Fig. 2005). albicans responds to farnesol. farnesol concentration. in part. 2. 1). This point is significant because such a study could only measure stable long-term farnesol-dependent changes in . In these cases the total farnesol levels are actually much higher than the added farnesol because the mutants themselves produce elevated levels of farnesol (Table 1. 2005. 2005) were done with mixed cell populations that differed in their ability to respond to farnesol. and timing were all important considerations for our experimental design. 2) corresponds with the commitment point. the effect of farnesol addition on the global gene expression during biofilm formation was determined at a single time point. We hypothesize that some of these changes are mediated by changes in the activity of the signaling pathways regulating morphogenesis. Enjalbert and Whiteway. beyond which added farnesol no longer blocks germ tube formation (Mosel et al. 2005. Further..68 Discussion C. 24 hours after addition of farnesol (Cao et al. see below).
in part. We also achieved a synchronous cell population by starting with resting cells and inoculating them in mGPP. 4).. we added farnesol at time zero in order to avoid commitment to filamentous growth. 2005). we have shown that nonsense-mediated mRNA decay (NMD) in S.. 2005). Two Tup1 co-regulators. Mitchell and Soll. Small changes in expression of a transcription regulator can have profound effects on the genes it regulates. while farnesol blocks the yeast to filament switch. but sufficient to affect expression of Adr1regulated genes. a transcription regulator of the genes responsible for making acetyl CoA and NADH from nonfermentable substrates. 1981. in these conditions we routinely get 95-100% filaments within 3-4 hrs. 1979. it is reasonable that farnesol regulates . Thus even though the change in Tup1 expression is relatively small it can have a profound effect on expression of the genes it regulates. It is relevant to farnesol’s mode of action because. Timing is also important because of the commitment phenomenon. were unaffected by farnesol at the mRNA level (Fig.69 gene expression. cerevisiae regulates accumulation of the mRNA for Adr1. The change in ADR1 mRNA levels is small (2. For example.. encoded by NRG1 and RFG1. to overexpression of Adr1 (Taylor et al. it does not block the elongation of preexisting filaments (Mosel et al.6-fold). the respiratory impairment seen in NMD mutants is due. and we harvested cells at 20 minute increments to observe changes in transcript levels during the early stages of the farnesol response (Mosel et al. Exposing a synchronized cell population to farnesol allowed us to detect subtle and consistent changes in transcript abundance.. In particular. Thus for our experiments. 2005). In this regard. This is the point at which a switch in the environmental stimulus no longer causes the expected switch in morphology (Chaffin and Wheeler. Mosel et al. 2005).
1987). albicans grown on the same plate (Fig.70 only one part of the complex. Because the tup1/tup1 and nrg1/nrg1 mutants did not respond to farnesol. for Ca2+ and calmodulin where only the Ca2+-calmodulin complex is active (Soto et al. DPP3 mRNA levels were not significantly elevated in the whole genome profiles of tup1/tup1 or nrg1/nrg1 mutants (Kadosh and Johnson.e.. 2007a). This regulation may be direct or indirect. 2005). however. 2A) is smaller than that reported for farnesol blockage of biofilm development. The enzyme responsible for ca. the tup1/tup1 and nrg1/nrg1 mutants of cytoplasmic Ca2+ overproduced farnesol while the rfg1/rfg1 mutant produced only slightly elevated farnesol (Table 1).. The rfg1/rfg1 mutant responded to farnesol. The increased TUP1 expression we observed for farnesol blockage of filament development (Fig. it suggests that farnesol acts through a pathway requiring Tup1 and Nrg1. This tup1/tup1 mutant overproduction is biologically significant because the excess farnesol produced by the tup1/tup1 mutant inhibits filamentation of wild-type C. Furthermore. that farnesol elevates TUP1 mRNA but not NRG1 or RFG1 mRNA. ca 6. indicating that the genes regulated by Rfg1 are not required for the response to farnesol. i... DPP3 does have a putative Nrg1 binding site in its promoter region. and that Nrg1 may work in concert with Tup1 to negatively regulate farnesol synthesis. 90% of farnesol synthesis is Dpp3 (Navarathna et al. 2005). The .6-fold as determined with DNA arrays (Cao et al. The juxtaposition between farnesol non-responsive mutants and the overproduction of farnesol implies that a farnesol-Tup1 feedback loop may exist. By analogy. fungi have the calmodulin in excess and regulate the activity of the complex by regulating the availability (Muthukumar et al. 2004a). 5).
at this time we can not exclude the possibility that post translational regulation of Efg1 is affected by farnesol... Efg1 is a transcriptional factor that activates hyphal gene expression including HWP1 and RBT1. 2004).. 4). Soto et al (Soto et al. CHK1 was elevated 6. EFG1 mRNA levels are regulated during filamentation. biofilm growth conditions as well as the fact that Cao et al.. 2005) used one time point 24 hours after farnesol addition. Additionally. these findings indicate that Tup1 is involved in mediating the C. 2004b) is consistent with a secondary effect of farnesol on Tup1. Both are downregulated by Tup1 and thus their downregulation by farnesol (Soto et al.71 difference in the response intensity may reflect filamentous vs. 2004b) suggested that farnesol acts by causing decreased CPH1 and HST7 mRNA levels. 2005). but they were not affected by farnesol since the timing and magnitude of the changes were similar in the presence and absence of farnesol (Fig. is also a Tup1 repressed gene. this suggests that farnesol does not regulate EFG1 mRNA levels but. 2004b) who also found no change in EFG1 mRNA levels at a single time point with added farnesol (Soto et al. albicans response to farnesol. These results are consistent with those of Soto et al (Soto et al. CPH1 is a transcription factor that regulates filamentous growth and HST7 is a MAP kinase kinase involved in filamentous growth. 2004b). Together with our results. . Chk1. Taken together. a histidine kinase shown to be required for the farnesol response (Kruppa et al.5-fold in the tup1/tup1 mutant (Kadosh and Johnson. Two other farnesol-related findings regarding filamentous growth can be accommodated in a Tup1-dependent model because they are downstream from Tup1.. (Cao et al..
We also thank Jessica A. Wiles for assisting with the germ tube assays and RNA work. and do not necessarily reflect the views of the National Science Foundation. University of Nebraska Foundation. albicans strains and the Tup1 antibody. and the Farnesol and Candida albicans Research Fund. This work was supported by grants from the National Science Foundation (MCB0110999). Any opinions. findings. conclusions.72 Acknowledgements We thank Alexander Johnson and Richard Cannon for providing us with C. or recommendations expressed in this report are ours. . the University of Nebraska Tobacco Settlement Biomedical Research Enhancement Fund.
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40. Act1 levels were used as a loading control. . 60 and 80 minutes post inoculation. tup1/tup1(BCa2-9).8) medium at 37°C in the presence or absence of 20 µM farnesol and incubated at 37°C for 60 minutes.8) medium at 37°C in the presence or absence of 20 µM farnesol. and a plot of average mRNA levels from a minimum of three independent experiments. TUP1 mRNA levels increased. The average fold Tup1 protein accumulation for farnesol treated cells relative to untreated cells is shown. CAI4. while two Tup1-regulated genes. albicans SC5314 resting cells were inoculated into mGPP (pH 4. 20. C. Tup1 protein levels are higher in the presence of farnesol. CAF2. rfg1/rfg (DU129).80 Figure Legends Figure 3-1. SC5314. ACT1 mRNA levels were used as a loading control.8) in the presence or absence of 20 μM farnesol and incubated at 37°C. HWP1 and RBT1 were downregulated in the presence of farnesol. Northern blots were prepared with total RNA from cells incubated both in the presence or absence of farnesol. tup1/tup1 (BCa2-10) and TUP1/tup1 (BCa2-3) resting cells were inoculated to mGPP (pH 4. Figure 3-3. albicans in conditions that promote germ tube formation and hyphal growth. Shown is a PhosphorImage of a representative Northern blot probed with radiolabeled TUP1 DNA (A). Cells were then harvested at 0. HWP1 DNA (B) and RBT1 DNA (C). Total protein extracts were prepared from SC5314 and TUP1/tup1 (BCa2-3) resting cells inoculated to mGPP (pH 4. Scale bar = 10µm Figure 3-2. and their cell morphology examined at 4 hours. nrg1/nrg1 (DU152). Response to farnesol by C.
The results are an average of three independent experiments. Farnesol does not affect expression of RFG1 or NRG1. RFG1. Resting cells were grown at 37 °C for 24 hours on YPD agar plates to allow for farnesol accumulation in the agar (horizontal streak.81 Figure 3-4. Figure 3-5. D) resting cells was plated (vertical streak) and incubated at 37 °C for an additional 24 hours. C. B) are zones of filament inhibition (as evident by smooth morphology) resulting from the farnesol produced by the horizontally streaked strains. Overproduction of farnesol by the tup1/tup1 mutant inhibits SC5314 filamentation. which encode DNA binding proteins that function with Tup1. Filamentation gives the wrinkled colony morphology seen below the arrows. NRG1. D)). scale bar = 10μm). which encodes a transcription activator of hypal-specific genes. either SC5314 (A.5X so that the colony morphology can be seen more clearly (center panels A. . or tup1/tup1 (BCa2-10. Micrographs of individual cells from the two bracketed regions are shown in the right panels (A.albicans strain SC5314 (B. C). The cells from the smooth regions are mainly yeasts. B) or tup1/tup1(C. The pictures in the two white boxes have been magnified 2. Area above the two arrows (left panels A. and EFG1 mRNA levels in SC5314 at 60 min after inoculation of resting cells into conditions that promote germ tube formation and hyphal growth in the presence and absence of 20μM farnesol. A. B. B). while there is a much larger proportion of filaments in the wrinkled region. or EFG1. Quantitative Northern blot analysis was used to measure TUP1. Subsequently.
82 Figure 3-1 .
83 Figure 3-2 .
84 Figure 3-3 .
85 Figure 3-4 .
86 Figure 3-5 .
8.6 ± 6. albicans strain (CAI-4) (CAF-2) tup1/tup1(BCa2-10) nrg1/nrg1 (DU152) rfg1/rfg1 (DU129) Farnesol responsea/ Positive Positive Negative Negative Positive Farnesol productionb/ 1. tup1/tup1 and nrg1/nrg1 null mutants do not respond to farnesol but overproduce farnesol. C. A negative response to farnesol indicates rough colony morphology in the presence and absence of farnesol.5 ± 12.0 ± 1.6 ± 0.2 4. .30 30.40 34. A positive response to farnesol indicates smooth colony morphology (yeast cells) in the presence of farnesol and rough colony morphology (filamentous cells) without added farnesol. the average value for CAI-4 and CAF-2 strains.87 Table 3-1.0 17 19 2.36 2. b/ c/ Farnesol production (μg/g dry weight of cells) was the average of three measurements Values based on fold increase over 1.6 Fold increase in farnesolc/ a/ Farnesol responses on GPP agar with and without 20μM farnesol incubated at 37oC for 48 hours.8 ± 2.
Cells were plated on a cornmeal agar plus Tween 80 plate under a coverslip and grown at 25°C for 25 hours. albicans Strain Cell morphology at the colony periphery No Farnesola/ Wild type (SC5314) TUP1/tup1 (BCa2-3) tup1/tup1 (BCa10) Yeasts + few filaments Yeasts + filaments filaments 20 μM Farnesol Yeast + few filaments Yeast + few filaments filaments a/ The phenotype for these strains grown on a cornmeal agar plus Tween 80 plate under a coverslip without farnesol was also previously reported (Braun and Johnson. .88 Table 3-2. C. 1997). Farnesol suppresses the haploinsufficient phenotype of a TUP1/tup1 heterozygote.
89 CHAPTER 4 A novel role for Czf1 in farnesol tolerance and the morphological response to farnesol in Candida albicans .
albicans to respond to farnesol in embedded conditions. and farnesol production levels were also examined. resulting in filamentous growth obstruction. but a clear understanding of the downstream effectors and signaling networks vital for the morphological farnesol response have yet to be unraveled. albicans. Finally. Czf1 was required for a wild-type farnesol response. farnesol toxicity. The quorum sensing phenomenon in Candida albicans is a process that inhibits the transition to filamentous growth and may be directly related to its ability to cause both mucosal and systemic disease. Here. CZF1 ectopic expression restored farnesol response in strains lacking Czf1. .90 Abstract Candida albicans is the primary cause of hospital acquired fungal infections in the United States. To address this issue. The Ras1-cyclic AMP signal transduction pathway is a proposed target for farnesol inhibition. highlighting a new role for Czf1 as a critical downstream effector of the morphological response to farnesol in C. we demonstrate for the first time the capability of C. The CZF1 mRNA transcript levels in response to farnesol. we screened a mutant library for farnesol resistant mutants. among the resistant mutants was a czf1Δ/czf1Δ mutant. and in these and liquid assays.
log phase cells that are energy-deprived are particularly sensitive. All of these cell types are affected by the quorum sensing molecule. Farnesol was the first eukaryotic quorum sensing molecule identified and was initially shown to block the transition from yeast to filaments (Hornby et al. Shirtliff et al. Saville et al.. For example. albicans morphology. White cells can also be killed by farnesol in the right environmental conditions. A polymorphic fungus. albicans can switch between yeast and filamentous forms of growth. 2001). 2009. while stationary phase cells in growth medium are quite farnesol tolerant (Langford et al.91 Introduction Candida albicans is an opportunistic pathogen that is part of the normal flora but can cause mild to severe opportunistic infections in immunocompromised patients.. thereby regulating opaque cell stability (Dumitru et al. 2002) and a protective role against oxidative stress (Deveau et al. albicans include mating-competent opaque cells and structures with unknown function called chlamydospores.. and low levels of farnesol induces necrosis in opaque cells when oxygen is present. 1997. C. 2010.. Subsequent studies identified farnesol as having an inhibitory role in biofilm formation (Ramage et al. farnesol. 2005). Uppuluri et al... very high levels of farnesol can increase chlamydospore formation (Martin et al. Two additional growth morphologies in C.. highlighting its influential role in C... 2007).. 2010. Westwater et al. and this transition is necessary for causing disease in a mouse model of infection (Lo et al. Given its important role in physiology.. 2005). it comes as no surprise that in vivo studies provide physiological relevance for farnesol signaling during infection and suggest farnesol plays distinct roles at different sites of infection. In addition. farnesol is a virulence factor in a . 2007). 2003).
2007a). 2008). Noffz et al. biofilm formation (Stichternoth and Ernst. 2008). Known roles of Czf1 include induction of contact-induced filamentous growth (Brown et al. In this paper. Chk1 (Kruppa et al. we have identified Czf1 (C. since no CZF1 mRNA was detected in an efg1Δ/ efg1Δ background (Vinces et al. 2009). Factors identified that play a role in the C. regulation between Efg1 and Czf1 may also occur at the protein level as well since these two proteins interact during a yeast two hybrid assay (Giusani et al.. 2006). 2006). albicans. Of note.. albicans zinc finger) as an important factor in the response to farnesol by screening a mutant library for mutants with a defective farnesol response.. Here we have defined a new role for Czf1 in the quorum sensing response of C.. 2008). the Hog1 MAP kinase pathway (Smith et al. the Cek1 MAP kinase pathway (Roman et al.. 2009). It contains an unusually large 5’ untranslated region (UTR) of approximately 2 kb (Vinces et al. albicans farnesol response include Tup1/Nrg1 (Kebaara et al. 2006). 2004). 2008).. 2002. 2007).. We show that Czf1 is not only necessary for a wild type morphological .... albicans.. yet it protects mice from oral candidiasis (Hisajima et al. These results highlight the need for a complete understanding of the signaling response induced by farnesol in C. Czf1 also has ties to the cAMP pathway.. Efg1. 1999). as a transcription factor downstream of cAMP signaling. Noffz et al.92 disseminated mouse model of infection (Navarathna et al. and can negatively regulate its own mRNA expression (Vinces et al. and white to opaque cell switching (Vinces and Kumamoto. This positive regulation by Efg1 is intriguing because Czf1 and Efg1 appear to play opposing roles in the cell with respect to morphology (Giusani et al... 2002. can bind the promoter of CZF1 and tightly regulate its expression. and the Ras1-cyclic AMP (cAMP) signaling pathway (Davis-Hanna et al. 2008).. 2004).
is required for farnesol tolerance as well. Our data suggest a vital downstream role for Czf1 in the signaling cascade elicited by farnesol in C. but in combination with Efg1. . albicans.93 response to farnesol in a variety of conditions.
Louis. For liquid farnesol response assays.8). Plates were incubated at 37°C for 2 days before colony morphology was assessed and compared to the parental strain. modified glucose phosphate proline (mGPP) pH 6.5 mM N-acetylglucosamine (GlcNAc) and uridine (40 µg/ml). A positive response to farnesol .8 was prepared as described in Kebaara et al (Kebaara et al. 2008) with 2. 0. 10.5% peptone. Resting cells were prepared as in Kebaara et al (Kebaara et al. and stored at 4°C overnight before use. MO) was stored under nitrogen and freshly prepared as a 100 mM stock solution in methanol for each experiment.. resuspended in potassium phosphate buffer.. or 50 µM farnesol. Yeast peptone dextrose (YPD) medium contained 1% yeast extract. Trans-trans farnesol (Sigma. and 2% dextrose. The defined medium. 2008) with modifications: single colonies were grown in 25 mL YPD broth at 30°C (unless otherwise noted) for 22-24h to reach stationary phase.94 Methods Strains and media Candida albicans strains and plasmids are listed in Table 1. and histidine added) agar plates containing 0. and solid medium included 2% agar. washed three times with 50 mM potassium phosphate buffer (pH6. Mutant library screen Mutants were obtained in 96-well plates and plated on mGPP (with 40 µg/ml uridine. Wrinkled/hairy colony morphologies were considered to be composed primarily of filaments and smooth colony morphologies were considered to be mostly yeast cells. BWP17 for farnesol response. shaking in glass flasks for the indicated times. Cells were then grown at 37ºC. arginine. resting cells were inoculated at 106 cells/mL in mGPP or mSPP (2% sucrose replacing glucose) broth with indicated farnesol concentrations. St.
2009). restriction digestion and Southern blotting were performed as described . CKY283 was plated on 5-fluoroorotic acid (5-FOA) containing media to select for ura. DNA analysis and transformation To create strain AAC2. Embedded cell growth Embedded media were prepared by mixing 104 cells/mL in 30 ml GPP (no GlcNAc added) or SPP molten agar (cooled to 50°C) with appropriate concentrations of farnesol and plated.95 was viewed as filamentous colonies without farnesol and smooth colonies in the presence of farnesol. even in the presence of 50 µM farnesol.mutants. 2002). Transformations were performed by the lithium acetate method and transformants were selected on media lacking uridine (Gietz and Woods.. Embedded micrographs were taken using a custom MVI TDM400 tetrad dissecting microscope and Sony Cybershot camera. Only colonies beneath the agar surface were examined. Microscopy and cell death determination Cellular morphology during the germ tube assays was determined using a Zeiss Stemi 2000-C light microscope. Cell death was determined by methylene blue staining as described by Gibson et al (Gibson et al. Newly created strains were confirmed by PCR and Southern blot analysis (data not shown). Embedded plates were incubated at 37°C for 12 or 17 hours as indicated. DIC micrographs were taken using an Olympus BX51 microscope and a Photometrics CoolSnap HQ CCD camera. AAC2 was subsequently transformed with BsgI- digested pDB212 to create strain AAC6. Farnesol resistant mutants maintained a filamentous morphology.
2005). MA). Real-time PCR reactions were carried out in an ABI Prism 7500 Real-Time PCR machine (Applied Biosystems). or 100 µM farnesol was added to each flask. and 0. 2001). Foster City. For harvesting cells.. . and mRNA integrity was assessed by measuring OD260/280 ratios and by examining the appearance on agarose gels. as described by Hornby et al (Hornby et al. Data shown are from a representative independent experiment performed in triplicate. 60. CA).96 in the GeneScreen Plus Hybridization Transfer and Detection Protocols (DuPont NEN Research Products. Farnesol Production Measurements Extracellular farnesol was extracted from cell-free supernatants of stationary phase cultures grown in GPP at 30ºC and analyzed by gas chromatography-mass spectrometry. 50. or 80 minutes. Quantitative real.time PCR Resting cells were inoculated into 75 mL mGPP broth at 5 x 106 cells/mL.. Extraction of mRNA was performed with the RiboPure Yeast Kit (Applied Biosystems. Cells were incubated at 37°C and harvested at 40. Boston. ACT1 mRNA levels were used as controls. Primers used to amplify CZF1 and ACT1 and PCR efficiencies were as previously described (Bassilana et al. Reverse transcription and PCR amplification were performed according to manufacturer’s specifications using MultiScribe Reverse Transcriptase and Power SYBR Green PCR Master Mix (Applied Biosystems). cultures were passed through glass fiber filters and cells were scraped off the filters to reduce the loss of filamentous cells during centrifugation.
2006).97 Results Identification of new genes required for the response to farnesol To identify important regulators of the farnesol response in C. respectively) were not examined further in this study to rule out mutants that are merely defective in morphological switching and not the specific response to farnesol. and they are summarized in Table 1. nor were any detected that demonstrated growth inhibition in the presence of farnesol. white/opaque cell switching. Mutants that were unable to interconvert between yeast and filaments (at 30° and 37°. Eight mutants were confirmed as farnesol resistant. CZF1 is required for a wild type morphological response to farnesol in both liquid and embedded conditions The czf1/czf1 mutant was identified as farnesol-resistant in the mutant library screen (Table 1) and selected for further characterization based on its known roles in morphogenesis. albicans. therefore it is desirable to obtain knockout mutants for further characterization due to the possibility of partial loss-of-function mutations. To test the role of Czf1 in the morphological response to farnesol. a transposon insertion mutant library of approximately 560 mutants (Supp. Mutants that were identified as farnesol resistant in the original screen were re-streaked on individual plates to rule out interference from neighboring mutant colonies and to confirm the farnesol resistant phenotype.. No mutants were identified that were hypersensitive to farnesol. Table 1) was screened for mutants defective in the morphological response to farnesol. a double null . and its connection to the cAMP pathway. The majority of mutants in this collection are Tn7-UAU1 plasmid insertion mutants (Nobile et al.
. and a haploinsufficient farnesol response phenotype was observed for the heterozygous mutant. we utilized defined GPP or SPP agar plates (lacking GlcNAc. For consistency with our prior work studying farnesol signaling and physiological significance (plates were incubated at 37°C). In both glucose and sucrose-containing media. while there was only a minimal farnesol response in non-inducing conditions. and the double null mutant ectopically expressing CZF1 under the control of the MAL2 promoter in a liquid farnesol response assay (Fig. 1999). the czf1Δ/czf1Δ mutant showed only a minimal reduction of germ tubes. glucose-containing media). SC5314 and CKY101 displayed the expected reduction of early stage filaments (germ tubes) when in the presence of farnesol. similar to that of the czf1Δ/czf1Δ mutant (Fig. These results show that the presence of Czf1 is critical to the ability of C. albicans to respond to farnesol in liquid media. 1B. albicans could respond to farnesol under such conditions and 2) Czf1 was needed for farnesol response when embedded in a matrix.98 czf1Δ/czf1Δ mutant was compared with its parental strain (CKY101). the heterozygous mutant. Results were similar whether glucose or sucrose was used as a carbon source. a wild type clinical isolate (SC5314). 1A. even in the presence of 100 μM farnesol. Since the first described role for Czf1 was to promote filamentation in embedded conditions (Brown et al. 1999).. However. sucrose-containing media). 1). . see experimental procedures) in our embedded assays. Although the growth media and incubation time and temperature used in this study are different from those initially used by Brown et al (Brown et al. we sought to determine whether: 1) C. Ectopic complementation of the czf1Δ/czf1Δ mutant was able to restore the farnesol response similar to that of the heterozygous mutant in pMAL2-inducing conditions (Fig.
the overall level of growth was more similar to that of wild type strains. however. As with the liquid farnesol response assays. respectively. possibly resulting in the expression of CZF1 and complementation of the farnesol response in both GPP and SPP embedded media. Ectopic CZF1 complementation of the czf1Δ/czf1Δ mutant produced positive farnesol responses similar to that of the heterozygote in both GPP and SPP. albicans to respond to farnesol in an embedded assay. 2). filamentous colonies were not observed. This demonstrates the capability of C. These . the czf1Δ/CZF1 heterozygote maintained a haploinsufficient phenotype in both farnesol treated and untreated samples: in untreated samples.99 we still observed a strong filamentation response from C. When SC5314 and CKY101 cells were grown in embedded media containing 50 μM farnesol. the MAL2 promoter (Fig. albicans SC5314 and CKY101 cells in response to growth embedded in a matrix (Fig. the czf1Δ/czf1Δ mutant exhibited a defective growth pattern in embedded conditions. 2). When the czf1Δ/czf1Δ mutant was grown in agar containing farnesol. and in farnesol-treated samples. filamentation was still observed. conditions which should turn off and on. 2). but far fewer colonies were present compared with the wild type and parental strains and colony morphology appeared different as well (Fig. As expected. As with the liquid germ tube formation assays. colonies were still filamentous. using glucose versus sucrose as a carbon source made no difference in filamentation or farnesol response (Fig. This suggests unintended leaky promoter activation in GPP media. colonies were reduced in hyphal formation but still more filamentous than wild type colonies treated with farnesol. 2). only a moderate farnesol response was observed in that the hyphae appeared shorter than in untreated samples.
CZF1 mRNA levels are decreased in the presence of farnesol Since CZF1 is regulated at the mRNA level. 3). These data indicate that CZF1 mRNA levels are decreased in the presence of farnesol. Mosel et al. new conditions in which to test C.100 results indicate that Czf1 is required for farnesol response in at least two distinct environmental conditions: liquid. 2008). A time-course experiment was performed to measure CZF1 expression levels in the presence of 0... during time points crucial to the farnesol response. showing a greater than 5fold decrease in expression relative to a sample lacking farnesol (Fig. 50 or 100 μM farnesol. 2008. albicans for a morphological farnesol response. aerobic growth media and embedded in a semi-solid matrix. Mutant cells grown in YPD broth at 30°C appeared small but elongated.. As with TUP1 mRNA levels (Kebaara et al. and 80 minute time points were selected based on previous studies showing the importance of this time frame to the farnesol response (Kebaara et al. an unusual temperature-regulated phenotype was observed. 2005). 3B). and 40. relative to samples containing no farnesol (Fig. CZF1 levels were most dramatically affected 60 minutes after treatment with farnesol. 60. At all 3 time points tested. both 50 and 100 μM farnesol reduced CZF1 levels. we asked whether CZF1 mRNA levels are affected by the presence of farnesol. reminiscent of . An efg1Δ/ efg1Δ czf1Δ/ czf1Δ double mutant exhibits decreased farnesol tolerance that is partially temperature dependent Upon growth of the efg1Δ/efg1Δ czf1Δ/czf1Δ mutant in liquid media for preparation of resting cells.
The parental strain CAI4 was used as a control in the 30°C resting cell group. and when the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant was grown in YPD broth at 37°C. the czf1Δ/czf1Δ mutant could not be tested accurately with methylene blue in these assays. The arguments for the viability of the czf1Δ/czf1Δ mutant in the presence of farnesol are twofold: 1) no growth inhibition was observed in the presence of farnesol on agar plates (data not shown) and 2) germ tube formation is a process that requires active protein synthesis (Imanishi et al. no significant cell death was observed in mGPP or in mSPP. albicans to survive and tolerate farnesol. 2004) and is unlikely to occur in a dead cell. For the cell death assays.. methylene blue staining was performed during liquid germ tube assays to more accurately assess the level of cell death occurring in these conditions (Fig. 4C-F). cells appeared larger and more rounded (Fig. CAI4 could also not be used as a control at 37°C for similar reasons. cells pre-grown at 37°C start as filaments. even in the presence of 100 μM farnesol.101 opaque cells (Fig. 2010). Given the importance of environmental conditions in the ability of C. except that cells were grown at either 30°C or 37°C in YPD broth until stationary phase was reached. When these efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant cells (pre-grown at 30°C) were tested for farnesol response in the previously described mGPP/mSPP liquid germ tube formation assays (experimental procedures). . due to its rapid formation of germ tubes which stain inconsistently with this dye regardless of the strain used or the presence of farnesol. On the other hand. 4C-F) were similarly resistant to farnesol-mediated killing in the liquid germ tube formation assays.. resting cells were prepared as described in experimental procedures. 4A). The czf1Δ/czf1Δ and efg1Δ/efg1Δ mutants (Fig. and as observed for SC5314 in Langford et al (Langford et al. we observed what appeared to be cell death. However. 4B).
102 the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant and the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant ectopically expressing CZF1 (from the 30°C resting cell group) showed extreme sensitivity to farnesol, regardless of the media used to induce CZF1 expression (Fig 4C, 4E). Conversely, when the efg1Δ/efg1Δ czf1Δ czf1Δ double mutant and the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant expressing CZF1 under the control of the MAL2 promoter from the 37°C resting cell group were tested for farnesol sensitivity, a general decrease in cell death was observed compared to the 30°C resting cell group (Fig. 4C-F). Based on these results, Czf1, Efg1, and temperature appear to play important roles in farnesol tolerance, and both 30°C and 37°C double mutant resting cells were subsequently utilized for additional experiments in this study.
Ectopically expressing CZF1 in an efg1Δ/ efg1Δ
partially restores filamentation and farnesol response in liquid and embedded conditions Can ectopic expression of CZF1 restore filamentation and response in the efg1Δ/ efg1Δ czf1Δ/czf1Δ double mutant? Liquid germ tube formation assays were performed on CAI4, efg1Δ/efg1Δ, the efg1Δ efg1Δ czf1Δ/czf1Δ double mutant, and the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant ectopically expressing CZF1 under the control of the MAL2 promoter, and only cells that did not stain with methylene blue were counted. For the 30°C resting cell group, only CAI4 was able to produce germ tubes and respond to farnesol, in both mGPP and mSPP (Fig. 5A, 5C). For the 37°C resting cell group, the efg1Δ/efg1Δ and the efg1Δ/ efg1Δ czf1Δ/czf1Δ double mutant were capable of producing very low levels of germ tubes with a slight trend of reduction in the presence of farnesol.
103 In conditions which induce CZF1 expression in the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant, an increase in germ tube formation was observed when compared to noninducing conditions (Fig. 5B, 5D), and farnesol reduced germ tube formation when CZF1 was expressed. When CAI4 30°C resting cells were inoculated into the embedded farnesol response assays, results were similar to SC5314 and CKY101 (Fig. 6). CAI4 produced filamentous colonies in the absence of farnesol in both GPP and SPP embedded media and the presence of farnesol resulted in decreased filamentation. Note the smaller hyphae growing out from the CAI4 colonies treated with farnesol (Fig. 6); because farnesol delays filamentation rather than block it entirely, the time points selected for observation are critical. Ideally CAI4 embedded plates should have been checked at 12 h postinoculation for farnesol response (as in Fig. 2) rather than at 17h as shown (no short filaments are observed in the farnesol-treated CAI4 samples at 12 h, data not shown), but the 17 h time point was selected in order to be consistent with the other slower-growing mutant strains being tested in Fig. 6. The efg1Δ/efg1Δ mutant was capable of forming filamentous colonies under embedded conditions, and a very subtle shortening of filaments was observed in farnesol-treated samples. An inoculum using 30°C resting cells for the efg1Δ/efg1Δ mutant is shown in Fig. 6, and similar results were obtained using an inoculum of 37°C resting efg1Δ/efg1Δ cells. The efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant was capable of producing filamentous colonies in the absence of farnesol, regardless of the temperature used during resting cell preparation (Fig. 6). The
efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant and the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant ectopically expressing CZF1 did not produce any colonies in the presence of farnesol
104 when 30°C resting cells were used (data not shown), suggesting these cells were killed by farnesol as they were in the liquid assays. When 37°C resting cells were used for the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant, farnesol reduced the number of colonies, but the colonies that grew were filamentous. Unexpectedly, the filamentous colonies from
farnesol-treated plates often grew in a different conformation from untreated samples. The efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant expressing CZF1 under the control of the MAL2 promoter behaved similarly to the efg1Δ/efg1Δ czf1Δ/ czf1Δ double mutant in noninducing conditions (Fig. 6A), and a subtle increase in colony size and filamentation was observed in inducing media (Fig. 6B). In the presence of farnesol, these filaments were slightly shorter. Taken together, both Czf1 and Efg1 are important for farnesol tolerance in embedded conditions as well, along with temperature. When CZF1 is ectopically expressed in a strain lacking Czf1 and Efg1, the level of filamentation is partially restored in both liquid and embedded assays, and farnesol partially suppresses filamentation in both conditions.
Overproduction of farnesol is not a general quality of farnesol-resistant mutants Two of the factors known to play a role in the C. albicans morphological farnesol response, Tup1 and Nrg1, both produced 17 and 19-fold higher levels of farnesol than wild type and parental strains (Kebaara et al., 2008). In order to determine whether farnesol overproduction is a general quality of farnesol-resistant mutants, we tested the farnesol production levels of the czf1Δ/czf1Δ mutant (Fig. 7). Farnesol production levels were not significantly altered in the czf1Δ/czf1Δ mutant, suggesting a more specific
105 involvement of Tup1/Nrg1 in farnesol production that does not require the presence of Czf1. .
is also mediated by the Ras1-cAMP pathway. 2008. Further evidence for the significant role of cAMP signaling during the farnesol response is provided by the identification of one of the protein kinase A (PKA) isoforms in C.. we identified new factors involved in the C. A recent study (Deveau et al. Czf1 does not appear to regulate farnesol production levels in the cell. was also shown to play an important part in farnesol tolerance. was not identified by the farnesol resistance screen as it was not present in this mutant library collection. albicans white a/α cells to survive in the presence of farnesol.. 2010). these are the first specific genes identified that control the ability of C. Tpk1. Here we showed that Czf1 is an important factor in the farnesol response. The Ras1-Cyr1-cAMP signaling pathway has been proposed to be the only direct target for farnesol in C. albicans. Surprisingly. Tpk2. It has a tight regulatory relationship with Efg1. Furthermore. albicans. Deveau et al. which is consistent with the Ras1-cAMP signaling pathway as a central target for farnesol. along with EFG1. The other PKA isoform. albicans (Davis-Hanna et al. 2010) showed that an additional phenotype of the farnesol response in C. as farnesol resistant. CZF1. The . reactive oxygen species (ROS) protection. albicans response to farnesol and have shown that Czf1 in particular plays a prominent role in the morphological response to farnesol in both liquid aerobic conditions and embedded conditions. this adds a new function for Czf1 during the quorum sensing response.. CZF1 mRNA levels are decreased in the presence of farnesol at critical time points during the farnesol response. This study provides additional evidence supporting a primary role of this pathway in farnesol signaling.106 Discussion In this study. and unlike another factor involved in farnesol signaling (Tup1).
2008. 2007) since opaque cells are inherently more sensitive to farnesol killing than are white cells (Dumitru et al. and it appeared to do the same in embedded media due to the lack of growth when using 30°C resting cell inocula. We speculate that it is no coincidence that both of these factors also play critical roles in white/opaque cell switching (Ramirez-Zavala et al.107 Hog1 signaling pathway was shown to participate in ROS protection as well. 2000. to provide protection from farnesol induced cell death. Czf1 and Efg1 have a synthetic effect on farnesol resistance.. while the double mutant is overly sensitive. YCK2. This study provides a new understanding of farnesol tolerance. It is intriguing that farnesol can protect the cell from ROS stress. albicans is potentially an active process (Langford et al. 1999. These mutants can possibly be used to fill in some of the gaps between these pathways... and HAP43.. Sonneborn et al.. energy-deprived white cells. The mere presence of CZF1 in the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant (via expression of CZF1 by the MAL2 promoter) was not sufficient. . 2007... 2007). however. Vinces and Kumamoto. as farnesol tolerance was only observed in the single mutants and not the double mutant. also function in different stress responses. Zordan et al. 2010). it is of interest to understand the key components that are involved. Three additional genes were identified that are potentially important for farnesol resistance: RLM1. Farnesol killed efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant cells in liquid aerobic conditions. Srikantha et al. while it can also kill opaque cells and log-phase. but through an unknown connection between the two pathways (Deveau et al. It is unclear why single efg1Δ/efg1Δ and czf1Δ/czf1Δ mutants are resistant to farnesol killing. Since farnesol tolerance in C. by identifying two factors that are required for survival in the presence of farnesol: Czf1 and Efg1. 2010).
in efg1Δ/efg1Δ czf1Δ/czf1Δ double mutants. Further. Through the course of testing farnesol response in conditions which either turn on or off the MAL2 promoter. farnesol was unable to suppress filamentation. CZF1 ectopic expression was able to partially restore filamentation and the morphological response to farnesol. 2007). there is likely some additional factor(s) that plays a minor role in farnesol tolerance and is regulated by shifts in temperature. It is also possible that the presence of Czf1’s native promoter is required for proper regulation in response to this particular stimulus when Efg1 is absent. Wor1/2 are potential candidates for such a role. 2006. Another intriguing question raised by this study is the role of temperature in farnesol tolerance... 2001).108 suggesting CZF1 may act upstream of EFG1 in this example. In cells lacking Czf1 (czf1Δ/czf1Δ mutants). Why was the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant more tolerant of farnesol when grown at 37°C? The answer may be tied to indirect farnesol production measurements suggesting that C.. While Czf1 was shown to be part of a farnesol-resistance mechanism in C. albicans produces slightly higher levels of farnesol at higher temperatures (Hornby et al. we have shown that the use of glucose versus sucrose as a carbon source had no distinguishable effect on the ability of the cells to respond to farnesol. Furthermore. These results are consistent with Czf1 functioning downstream of Efg1 during the morphological farnesol response. in part due to their regulation of white/opaque switching (Huang et al. Zordan et al. albicans. . Zordan et al. it also strongly contributes to the morphological response to farnesol. and ectopic complementation of CZF1 in these cells restored the ability of farnesol to block germ tube formation in both liquid and embedded environmental conditions.. 2006.
Lodderomyces guillermondii (data not shown). In conclusion. Furthermore.. As an example. 2009)].. Weber et al. but little is known about the regulation of Tup1/Nrg1 and how these factors fit into the farnesol signaling network. albicans tolerance to farnesol as well as farnesol-mediated filament inhibition. 2007. The finding that Czf1 plays a central role in the unique morphological and tolerance responses in C. albicans appear to be relatively unique to the fungus.109 The many effects of farnesol on C. 2006) while other Candida species contain CZF1 homologs. is apparent. 2008) has the highest homology to the C. For example. Furthermore. is farnesol tolerant. such as the cAMP pathway. albicans is consistent with the observation that Saccharomyces cerevisiae (which does not respond to farnesol) and other closely related ascomycetes lack a Czf1 homolog (Vinces et al.. While the elongisporius. cross-regulation among many of the farnesol response pathways was recently summarized (Deveau et al.. albicans CZF1 gene with 81% identity at the nucleotide level (data not shown). and morphologically responds to farnesol (Henriques et al. we have identified new roles for Czf1 in mediating the C. a Candida species that produces the second highest farnesol levels.. and Candida connection of Czf1 to other factors known to play a role in the C. the CZF1 gene in Candida dubliniensis. albicans farnesol response. Candida lusitaniae. 2006) appear to be conserved to varying degrees in additional Candida species whose sequence information has been recently made available including: Candida tropicalis. the unusually large 5’ upstream region of CZF1.. and neighboring genes at the CZF1 locus (Vinces et al. compared with other bacterial species and fungal genera [reviewed by Langford et al (Langford et al. others links remain unclear. other proteins . the CZF1 gene itself. 2010).
and G-actin (Hall and Muhlschlegel. 2009.). We would also like to thank Steve Harris and Gabriel Langford for use of their microscopes and cameras.). Zou et al.110 involved in Ras1-Cyr1-cAMP signaling such as Ras2. Cap1. . University of Nebraska Foundation (to K. Zhu et al. 2009. the HammondMaude Fling Fellowship (to M. and the Farnesol and Candida albicans Research Fund. Carol Kumamoto. Alexander Johnson.W. This work was supported by a faculty seed grant from the Constance Miriam and Ethel Corrine Syford Memorial Fund (to A.L.).A.L.L..N. and Gerald Fink for kindly providing the strains and plasmids used in this study.. Acknowledgements We are grateful to Aaron Mitchell. Dominique Sanglard. albicans by demonstrating the presence of new connections between signaling pathways. This points to the fact that farnesol can be an extremely useful tool for studying signal transduction in C. 2009) have not been tested for possible roles in farnesol signaling and may yet prove to be involved.
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C. Cells were . or 100 μM farnesol and subsequently harvested for RNA extraction. Data are averages from three replicates. czf1Δ/CZF1= CKY116. and incubated at 37°C for 12 h. CZF1 mRNA expression is decreased in the presence of farnesol. C. Figure 4-4. respectively) with 0. or 80 minutes (A-C. albicans resting cells were inoculated into glass flasks at 106 cells/mL in either mGPP (A) or mSPP (B) broth. SC5314 resting cells were inoculated into mGPP broth for 40. Czf1 is required for the morphological response to farnesol in embedded media. and temperature play a role in farnesol tolerance. Scale bar = 10 μm. Data shown are from independent experiments performed in triplicate. albicans resting cells were mixed with either GPP (A) or SPP (B) molten agar and 0 or 50 μM farnesol as described in experimental procedures. Czf1. The czf1Δ/ czf1Δ efg1Δ/ efg1Δ double mutant was grown in YPD broth at 30°C (A) or 37°C (B) for 24 h and differences cell morphology was observed. 60. 50. or 100 μM farnesol was added. these were repeated with similar results on at least two separate occasions. Figure 4-2. 50.119 Figure Legends Figure 4-1. Cultures were incubated for 1 h at 37°C with shaking at 225 rpm and percentage of germ tube formation was subsequently determined (czf1Δ/ czf1Δ = CKY230. and 0. Efg1. CZF1 levels in the minus farnesol samples were set at zero and fold change in CZF1 levels for samples with farnesol added are shown. Quantitative real-time PCR was used to measure relative CZF1 mRNA levels with ACT1 used as a reference gene. Figure 4-3. czf1Δ/ czf1Δ pMAL2-CZF1 = CKY231). Czf1 is required for the morphological response to farnesol in liquid conditions. Independent experiments were repeated in duplicate with similar results.
these were repeated with similar results on at least two separate occasions. Figure 4-5. Independent experiments were repeated in duplicate with similar results. C. Data shown are from independent experiments performed in triplicate. and incubated at 37°C for 17 h. 50.B) or mSPP (C. . albicans resting cells (prepared at 30°C or 37°C) were mixed with either GPP (A) or SPP (B) molten agar and 0 or 50 μM farnesol as described in experimental procedures. Cultures were incubated at 37°C with shaking at 225 rpm. Cells were grown at either 30°C or 37°C until stationary phase was reached to prepare resting cells. czf1Δ/czf1Δ efg1Δ/efg1Δ pMAL2-CZF1 = AAC6). Figure 4-6. 106 cells/mL of these resting cells were inoculated into mGPP (C.120 grown at either 30°C or 37°C in YPD broth until stationary phase was reached (24 h. Czf1 and Efg1 are both required for a wild type morphological response and tolerance to farnesol in embedded agar. and cell death by methylene blue staining was determined after 90 min (efg1Δ/ efg1Δ = HLC67. Data shown are from independent experiments performed in triplicate. Cultures were incubated at 37°C with shaking at 225 rpm. 106 cells/mL of these resting cells were inoculated into mGPP (A. except 48 h for CAI4 and efg1Δ/ efg1Δ strains) to prepare resting cells as described in experimental procedures. these were repeated with similar results on at least two separate occasions.D) broth and 0. 50.F) broth and 0. Colonies in figure were from 30°C resting cells unless otherwise noted. czf1Δ/czf1Δ efg1Δ/efg1Δ = CKY283. and percentage of germ tube formation was subsequently determined. or 100 μM farnesol was added. or 100 μM farnesol was added. Ectopic expression of CZF1 in a czf1Δ/czf1Δ efg1Δ/efg1Δ double mutant partially restores filamentation and farnesol response in liquid conditions.D) or mSPP (E.
. Cells were grown in GPP broth at 30°C for 48 hours prior to farnesol extraction and quantification.121 Figure 4-7. as described in methods. A czf1Δ/czf1Δ mutant produces farnesol levels similar to that of wild type and parental strains.
122 Figure 4-1. mSPP 100 80 % germ tube formation 60 0 FOH 50 μM FOH 100 μM FOH 40 20 0 SC5314 CKY101 czf1Δ/czf1Δ czf1Δ/CZF1 czf1Δ/czf1Δ pMAL2-CZF1 . Liquid Farnesol Response Assay. mGPP 100 80 % germ tube formation 60 0 FOH 50 μM FOH 100 μM FOH 40 20 0 SC5314 CKY101 czf1Δ/czf1Δ czf1Δ/CZF1 czf1Δ/czf1Δ pMAL2-CZF1 B. Liquid Farnesol Response Assay. A.
123 Figure 4-2 .
80 min Normalized Relative Fold Change in Expression 0 FOH 0 -2 -1. 40 min Normalized Relative Fold Change in Expression 0 FOH 0 -2 -4 -6 -8 -10 -2. CZF1 mRNA Levels.57 -4.41 50 µM FOH 100 µM FOH C.124 Figure 4-3 A. CZF1 mRNA Levels.23 -4 -6 -8 -10 -1.97 -10 -5.08 50 µM FOH 100 µM FOH B. CZF1 mRNA Levels.33 50 µM FOH 100 µM FOH == . 60 min Normalized Relative Fold Change in Expression 0 FOH 0 -2 -4 -6 -8 -5.
mGPP 80 60 % Cell Death 0 FOH 50 μM FOH 100 μM FOH 40 20 0 CAI4 efg1Δ/efg1Δ czf1Δ/czf1Δ czf1Δ/czf1 efg1Δ/efg1Δ Δefg1Δ/efg1Δ pMAL2-CZF1 D. C. Cells grown at 37°C. mGPP 80 60 % Cell Death 40 20 0 efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ pMAL2-CZF1 .125 Figure 4-4 A. Cells grown at 30°C. B.
Cells grown at 30°C. mSPP 80 60 % Cell Death 40 20 0 efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ pMAL2-CZF1 . Cells grown at 37°C.126 Figure 4-4 E. mSPP 80 60 % Cell Death 40 20 0 CAI4 efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ pMAL2-CZF1 F.
Cells grown at 37°C. mGPP 100 0 FOH B. mGPP 100 50 μM FOH 100 μM FOH 80 60 40 20 0 CAI4 % germ tube formation % germ tube formation 80 60 40 20 0 efg1Δ/efg1Δ czf1Δ/czf1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ efg1Δ/efg1Δ pMAL2-CZF1 efg1Δ/efg1Δ czf1Δ/czf1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ efg1Δ/efg1Δ pMAL2-CZF1 C.127 Figure 4-5 A. mSPP 100 % germ tube formation 80 60 40 20 0 efg1Δ/efg1Δ czf1Δ/czf1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ efg1Δ/efg1Δ pMAL2-CZF1 . Cells grown at 37°C. Cells grown at 30°C. mSPP 100 % germ tube formation 80 60 40 20 0 CAI4 efg1Δ/efg1Δ czf1Δ/czf1Δ czf1Δ/czf1Δ efg1Δ/efg1Δ efg1Δ/efg1Δ pMAL2-CZF1 D. Cells grown at 30°C.
128 Figure 4-6 .
5 0 SC 53 14 CA I4 .129 Figure 4-7 Farnesol Production 2 ug FOH/g dry wt.5 1 0. 1.
contributes to epithelial cell damage (Park et al. 2001. 2008. Summary of insertion mutants with an impaired farnesol response. Vinces and Kumamoto.... 2001. 2007) Catalytic subunit of cAMP-dependent protein kinase A... cerevisiae ortholog None Predicted or known biological processes Zinc finger transcription factor for filamentation under embedded conditions and positive regulator of white to opaque switching.. 2009) Putative transcription factor Transcription factor. Giacometti et al... 2009) Transcription factor for amino acid permease genes (Martinez and Ljungdahl. 2007. 2008) tpk1/tpk1 Tpk2 rlm1/rlm1 Rlm1 Stp2 Hof1 Yck2 Hap1 Yap3 stp2/stp2 hof1/hof1 yck2/yck2 zcf14/zcf14 hap43/hap43 . Maidan et al.. Hornby et al. 2006) Maintenance of cell polarity. Zordan et al.. 2005) Role in cytokinesis (Li et al. 2009. 2006. Cloutier et al. Vinces et al. 2005) Transcription factor for genes involved in cell wall organization and biogenesis and various stress responses (Bruno et al. 2006.130 Table 4-1. involved in multiple stress responses (Bockmuhl et al. Sampaio et al. 1999. Ramirez-Zavala et al..... regulator of morphogenesis. Mutant czf1/czf1 S. antimicrobial peptide resistance. Tpk2 isoform. binds Efg1 and expression controlled by Efg1 and Czf1 (Brown et al. involved in iron limitation response (Baek et al. 2003.
.. 1993) Aaron Mitchell (Nobile and Mitchell.. Candida albicans strains and plasmids used in this study. Parental strain C. 2002) This study This study AAC6 AAC2 (czf1Δ/ czf1Δ efg1Δ/ efg1Δ pMAL2-CZF1) Plasmids pDB212 Carol Kumamoto (Brown et al. 2001) Alexander Johnson (Fonzi and Irwin... 1999) Carol Kumamoto (Giusani et al. 2005) Gerald Fink (Lo et al. 1999) ... 1999) Carol Kumamoto (Vinces et al. 1984) Alexander Johnson (Leberer et al. albicans strains SC5314 CAF2 CAI-4 BWP17 HLC67 (efg1Δ/ efg1Δ) CKY101 CKY230 (czf1Δ/ czf1Δ) CKY116 (czf1Δ/CZF1) CKY231 (czf1Δ/ czf1Δ pMAL2-CZF1) CKY283 (czf1Δ/ czf1Δ efg1Δ/ efg1Δ) AAC2 Clinical isolate CAI-4 CAF2 RM1000 CAI-4 CAI-4 CAI-4 CAI-4 CAI-4 CAI-4 CKY283 Genotype/Description Source Clinical isolate Ura+ derivative of CAI-4 ura3::imm434/ura3::imm434 ura3::imm434/ura3::imm434 arg4::hisG/arg4::hisG his1::hisG/his1::hisG ura3::imm434/ura3::imm434 efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 ade2::pDB152 ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG ade2::pMAL2-URA3 ura3::imm434/ura3::imm434 CZF1/czf1::hisG -URA3-hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG ade2::pMAL2-CZF1-URA3 ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG-URA3hisG efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG efg1::hisG/efg1::hisG ura3::imm434/ura3::imm434 czf1::hisG/czf1::hisG efg1::hisG/efg1::hisG ade2::pMAL2-CZF1-URA3 pMAL2-CZF1 URA3 ade2’ Ampr Alexander Johnson (Gillum et al.131 Table 4-2.. 2006) Carol Kumamoto Carol Kumamoto (Brown et al. 1997) Carol Kumamoto (Brown et al.
1409 orf19.3208 orf19.856 orf19.9791 orf19.7389 orf19.4746 orf19.5235 orf19.4658 .11450 orf19.1795 orf19.4669 orf19.1857 orf19.6952 orf19. Mutant ORF# orf19.5011 orf19.7479 orf19.5328 orf19.7412 orf19. List of mutants screened in this study.11598 orf19.6760 orf19.8907 orf19.4369 orf19.6970 orf19.5037 orf19.9364 orf19.7291 orf19.3995 orf19.132 Supplementary Table 4-1.10248 orf19.3171 orf19.4966 orf19.564 orf19.4668 orf19.8635 orf19.9508 orf19.2723 orf19.14178 orf19.4284 orf19.9364 orf19.768 orf19.
4257 orf19.1805 orf19.5251 orf19.3012 orf19.2237 orf19.1509 orf19.12351 orf19.5445 orf19.3009 orf19.695 orf19.1593 orf19.13950 orf19.769 orf19.1825 orf19.3202 orf19.3014 orf19.9081 orf19.2237 orf19.3171 orf19.771 orf19.6261 orf19.10169 .3563 orf19.10359 orf19.4763 orf19.5571 orf19.6729 orf19.6036 orf19.3701 orf19.2033 orf19.3396 orf19.5495 orf19.11973 orf19.133 orf19.2990 orf19.5365 orf19.7381 orf19.580 orf19.5571 orf19.3764 orf19.12265 orf19.7614 orf19.
4369 orf19.2660 orf19.7194 orf19.6185 orf19.5094 orf19.9331 orf19.2763 orf19.134 orf19.4369 orf19.2392 orf19.2653 orf19.3906 orf19.8326 .9115 orf19.6194 orf19.2348 orf19.3818 orf19.5292 orf19.3678 orf19.8837 orf19.7400 orf19.4519 orf19.271 orf19.7201 orf19.1005 orf19.7016 orf19.4535 orf19.1759 orf19.7472 orf19.4457 orf19.4529 orf19.11598 orf19.4244 orf19.4428 orf19.658 orf19.6344 orf19.6032 orf19.12603 orf19.7389 orf19.4893 orf19.5001 orf19.1614 orf19.
13704 orf19.811 orf19.3530 orf19.4872 orf19.4979 orf19.7410 orf19.6950 orf19.6194 orf19.2938 orf19.530 orf19.4214 orf19.3125 orf19.1394 orf19.135 orf19.6737 orf19.14148 orf19.769 orf19.7208 orf19.7451 orf19.6100 orf19.5866 orf19.6850 orf19.7381 orf19.6971 orf19.4409 orf19.9115 orf19.4513 orf19.1252 orf19.3190 orf19.7207 orf19.12706 orf19.4285 orf19.2808 orf19.271 orf19.2270 orf19.2901 orf19.4440 orf19.7401 orf19.5352 orf19.6365 .1510 orf19.7400 orf19.
4518 orf19.7447 orf19.5761 orf19.7583 orf19.1795 orf19.2061 orf19.7610 orf19.4543 orf19.6365 orf19.7576 orf19.10841 orf19.6267 orf19.136 orf19.4116 orf19.1291 orf19.4969 orf19.1793 orf19.3122 orf19.11659 .7381 orf19.7324 orf19.425 orf19.3707 orf19.1907 orf19.4139 orf19.829 orf19.248 orf19.5887 orf19.813 orf19.5664 orf19.4243 orf19.3844 orf19.5100 orf19.4823 orf19.3254 orf19.4188 orf19.3678 orf19.4846 orf19.1628 orf19.2350 orf19.1331 orf19.1276 orf19.
11410 19.3809 19.4972 19.998 orf19.2745 19.6763 orf19.7207 orf19.7583 19.137 orf19.411 19.6817 19.7518 19.173 19.2508 orf19.4318 19.4433 19.5016 orf19.3012 orf19.5908 19.5729 19.7355 orf19.5026 19.12215 19.4010 orf19.814 orf19.4125 19.4743 orf19.6036 orf19.4670 19.5023 orf19.7570 .7451 orf19.7150 19.12786 19.4766 19.7359 19.3986 19.7247 19.723 19.3187 19.4474 orf19.6680 19.
798 19.5338 19.9191 19.4568 19.1497 19.1135 19.2647 19.1255 19.2356 19.2260 19.889 19.3305 19.5548 19.2393 19.13396 19.138 19.2612 19.1358 19.4573 19.6182 19.4778 19.1187 19.2674 19.1496 .2280 19.2458 19.2399 19.2054 19.2315 19.3088 19.7318 19.4767 19.2331 19.4776 19.1007 19.9326 19.909 19.3308 19.431 19.1178 19.3127 19.5380 19.
735 19.3407 19.139 19.3405 19.567 19.6781 19.299 orf19.3966 orf19.4649 19.4887 orf19.1694 19.3193 19.3390 19.1826 19.1563 .1565 19.2623 19.2432 19.1822 19.6845 19.2077 19.3683 19.976 orf19.6985 19.1589 19.10478 19.2064 19.1227 19.299 orf19.2623 orf19.3300 19.3835 19.487 19.7025 19.3753 19.3966 orf19.6850 19.4887 orf19.1729 19.718 19.10266 19.2623 orf19.
3869 orf19.3869 orf19.5412 orf19.5867 orf19.4884 orf19.3642 orf19.7251 orf19.2613 orf19.1 orf19.1490 orf19.2613 orf19.4981 orf19.5867 orf19.2476 orf19.1277 orf19.1 orf19.1563 orf19.3869 orf19.1714 orf19.1563 orf19.5674 orf19.1490 orf19.7374 orf19.1277 orf19.1 orf19.3010.3434 orf19.4884 orf19.140 orf19.4981 orf19.3434 orf19.4981 orf19.893 orf19.2476 orf19.3193 orf19.3188 .532 orf19.532 orf19.2647 orf19.1490 orf19.2476 orf19.2613 orf19.3010.166 orf19.3010.
173 orf19.681 orf19.5975 orf19.5133 orf19.3012 orf19.7371 orf19.4662 orf19.4766 orf19.217 orf19.6817 orf19.1358 orf19.6203 orf19.141 orf19.255 orf19.6121 orf19.2623 orf19.3294 orf19.5953 orf19.5848 orf19.6985 orf19.4649 orf19.6407 orf19.1032 orf19.2315 orf19.2356 orf19.3434 orf19.6102 orf19.1069 orf19.2748 .1035 orf19.2540 orf19.4961 orf19.2423 orf19.3190 orf19.6038 orf19.723 orf19.5917 orf19.1718 orf19.1499 orf19.861 orf19.6124 orf19.5849 orf19.
4084 orf19.5924 orf19.4084 orf19.469 .5181 orf19.4252 orf19.4145 orf19.2808 orf19.3252 orf19.6626 orf19.4252 orf19.4524 orf19.2395 orf19.4866 orf19.4225 orf19.4166 orf19.2217 orf19.7388 orf19.896 orf19.5940 orf19.5759 orf19.4866 orf19.3876 orf19.1813 orf19.7523 orf19.7388 orf19.6227 orf19.702 orf19.7319 orf19.4979 orf19.142 orf19.3405 orf19.4288 orf19.7372 orf19.7001 orf19.6713 orf19.5995 orf19.4450 orf19.5181 orf19.7001 orf19.896 orf19.2753 orf19.
794 orf19.663 orf19.5350 orf19.5408 orf19.143 orf19.835 orf19.4892 orf19.3256 orf19.469 orf19.35 .4892 orf19.223 orf19.4084 orf19.2605 orf19.2341 orf19.2605 orf19.3047 orf19.2395 orf19.5224 orf19.451 orf19.794 orf19.5224 orf19.3256 orf19.469 orf19.4242 orf19.1196 orf19.5350 orf19.1874 orf19.35 orf19.4084 orf19.5408 orf19.2395 orf19.1874 orf19.835 orf19.1196 orf19.3047 orf19.223 orf19.4242 orf19.663 orf19.469 orf19.2341 orf19.451 orf19.
4867 orf19.2678 orf19.7281 orf19.3545 orf19.4909 orf19.4909 orf19.3049 orf19.4269 orf19.4890 orf19.7044 orf19.4001 orf19.4308 orf19.5068 orf19.6913 orf19.7044 orf19.7510 orf19.5224 orf19.5357 orf19.6913 orf19.7510 orf19.5253 .4001 orf19.4432 orf19.7510 orf19.3545 orf19.3854 orf19.4347 orf19.4890 orf19.4308 orf19.7510 orf19.6369 orf19.3744 orf19.4867 orf19.5068 orf19.4347 orf19.3854 orf19.4144 orf19.7281 orf19.4432 orf19.144 orf19.3751 orf19.5224 orf19.
2277 orf19.3841 orf19.846 orf19.844 orf19.2102 orf19.3415 orf19.4297 orf19.3415 orf19.3841 orf19.2277 orf19.4297 orf19.145 orf19.428 orf19.4518 orf19.2268 orf19.1341 orf19.844 orf19.6889 orf19.7355 orf19.846 orf19.4518 orf19.3530 orf19.3049 orf19.7523 orf19.3720 orf19.6243 orf19.4002 orf19.3049 orf19.3415 orf19.3720 orf19.428 orf19.6889 .7451 orf19.7451 orf19.3530 orf19.2436 orf19.2910 orf19.3256 orf19.7523 orf19.3256 orf19.2910 orf19.5253 orf19.
7510 orf19.5162 orf19.7652 orf19.2222 orf19.5911 orf19.895 orf19.460 .1283 orf19.130 orf19.5901 orf19.1283 orf19.1341 orf19.1283 orf19.1341 orf19.146 orf19.7652 orf19.5325 orf19.5162 orf19.5911 orf19.7510 orf19.460 orf19.1283 orf19.7164 orf19.895 orf19.130 orf19.5325 orf19.7164 orf19.5901 orf19.
M. 4: 1353-62. (2009) Cellular Interactions of Farnesol. and Nickerson.147 CHAPTER 5 Summary and Future Directions With a brief excerpt from: Langford. .L. Future Microbiol.L.. a Quorum Sensing Molecule Produced by Candida albicans. Atkin. A.W. K..
It seems that farnesol suppression of filamentation during C. albicans has evolved mechanisms to produce and tolerate higher levels of farnesol and use it as a quorum sensing molecule. Farnesol Tolerance and Sensitivity in C. Conversely. A current challenge is to develop a unifying model for the mode of action for farnesol in eukaryotes and elucidate the mechanisms used by C. C. we have defined conditions that stimulate sensitivity to farnesol in C. Czf1 also played a dual role in the farnesol response. two novel factors were identified that are involved in the morphological response to farnesol: Tup1 and Czf1 (Chapters 3. and for many cell types. albicans white cell farnesol tolerance during quorum sensing remains unclear. albicans quorum sensing works through both positive and negative regulators of filamentation (Czf1 and Tup1. a low level of farnesol is sufficient to inhibit growth or induce cell death. respectively) to achieve the resulting change in morphology.148 Summary Farnesol has effects on all eukaryotic cells tested. albicans is able to resist farnesol-mediated cell death: stationary phase cells provided with an energy source. In this study. albicans to evade the adverse effects of farnesol and respond to farnesol as a signaling molecule. as it was partially responsible for farnesol tolerance in quorum sensing conditions. albicans: energy-deprived. albicans to tolerate farnesol and use it as a signaling molecule. we established environmental conditions in which C. Using the aforementioned environmental conditions that allow C. In contrast. Since the effects of farnesol on other organisms have . log phase cells (Chapter 2). albicans The precise mechanism for C. 4).
149 been studied in seventeen different species (summarized in Chapter 1), this is an important mechanism to understand in the field of interspecies communication. Furthermore, as exogenous farnesol has many effects in the mammalian host during infection such as preventing infection of mucosal surfaces, increasing virulence in a disseminated infection and interference of the host cytokine response [Chapter 1 and (Navarathna et al., 2007)], understanding the farnesol tolerance mechanism in C. albicans may prove useful therapeutically. It is unknown how C. albicans white cells are resistant to the toxic effects of farnesol. Do the cells have a type of passive resistance, such as changes in membrane composition, or do they undergo active resistance, such as conversion of farnesol into a non-toxic compound? In order to look for genes that may be involved in farnesol tolerance, a screening approach was used (Chapter 4). However, the screen was
unsuccessful in identifying mutants that were growth-inhibited (sensitive) in the presence of farnesol, even at levels as low as 10 µM. Even so, the mutant library collection used in this study is by no means a complete representation of the entire collection of C. albicans predicted open reading frames (ORFs); there are 6197 predicted ORFs in the genome, compared with the 560 mutants contained within the mutant library collection used, leaving plenty of room for future identification of farnesol tolerance factors as more mutants become available. The identification of Czf1 and Efg1 as important players in farnesol survival is intriguing because a farnesol-sensitive phenotype was observed only when both genes were deleted. Ectopic expression of CZF1 in the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant could not provide complete protection from farnesol killing yet the single efg1Δ/efg1Δ
150 and czf1Δ/czf1Δ mutants were unharmed (Chapter 4). Perhaps Czf1 functions upstream of Efg1 in this situation, or the normal regulation of CZF1 might be especially important in the absence of EFG1. In the reverse situation, could ectopic expression of EFG1 in the double mutant restore partial or full farnesol resistance? Czf1 and Efg1 likely regulate a redundant gene or set of genes for farnesol tolerance because the single mutants do not show a farnesol sensitive phenotype while the double mutant exhibits a synthetic farnesol sensitivity phenotype. While changes in gene expression have been extensively studied in the efg1Δ/efg1Δ mutant compared to wild type/parental strains (Braun and Johnson, 2000; Harcus et al., 2004; Sohn et al., 2003), there is no matching data for Czf1 available. Microarray analyses designed to complement existing data on Efg1 may reveal genes that are commonly regulated by Czf1 and Efg1, either directly or indirectly. Genes whose expression was altered in cells lacking Czf1 and Efg1 could also be compared to existing microarray data comparing gene expression between white and opaque cells (Lan et al., 2002; Tsong et al., 2003; Zhao et al., 2005). Genes that are differentially expressed in opaque cells, efg1Δ/efg1Δ, and czf1Δ/czf1Δ mutants would comprise a list of strong candidates for potential farnesol resistance factors. One further step that may be taken would be to look at other farnesol-sensitive organisms to see if homologs to the candidate farnesol resistance genes are present in those species. The role of temperature in farnesol tolerance (the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant was more sensitive at 30°C than at 37°C) may be correlated to evidence suggestive of slightly elevated farnesol production levels at higher temperatures (Hornby et al., 2001). It would be informative to examine changes in gene expression and to measure farnesol production in the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant at both 30°C
151 and 37°C, compared to wild type and parental strains. Differentially regulated genes may be partly responsible for the cellular morphology and farnesol resistance changes observed at these two temperatures. One would also predict that farnesol production may be significantly lowered at 30°C based on the degree of farnesol sensitivity for the double mutant cells grown at this temperature. Analysis of the efg1Δ/efg1Δ single mutant would need to be included in this study; initial results using this mutant resulted in high variation between replicates (M.L. Langford, data not shown) and further replicates will need to be conducted to determine whether Efg1 plays a role in farnesol production. Furthermore, a possible role for Tup1 in farnesol resistance should not be ignored. As tup1Δ/tup1Δ mutants produced 17-fold more farnesol compared to wild type and parental strains, these cells may have heightened abilities to tolerate farnesol. However, it will be difficult to study higher tolerance to farnesol in these mutant cells since they are locked in the filamentous morphology (unless secondary mutations are introduced), unlike the efg1Δ/efg1Δ and czf1Δ/czf1Δ mutants. Preliminary experiments must first be conducted to better understand the tup1Δ/tup1Δ farnesol resistance, such as a comparison of farnesol tolerance between yeast, pseudohyphal, and hyphal cells. If the loss of Tup1 results in higher farnesol tolerance, would a triple efg1Δ/czf1Δ/tup1Δ mutant have restored resistance? What would the farnesol production levels in that mutant be? It is intriguing that all three of these factors play a prominent role in white-opaque switching, farnesol production and/or farnesol tolerance, as well as the morphological response to farnesol.
2005). The ras1Δ/ras1Δ mutant is defective in filamentation under aerobic conditions and embedded in YPS (yeast extract. the morphological response to farnesol can be tested. specifically during embedded growth. Two models (network and central processor) have been proposed with regard to C. but it has not been tested in our GPP embedded media. 2000) and are worth considering when examining the farnesol response. If filamentation can be achieved in our embedded assay.. this would both complement . it remains a possibility that there may be multiple targets for farnesol in the cell. 2008. Deveau et al. sucrose) agar (Maidan et al.. That study suggested C. This conclusion was reached when they observed that several hyphal-specific genes are not regulated in unison in different hyphal inducing conditions but in fact respond as individuals. albicans follows the network signaling model. albicans Because the presence of a single farnesol receptor has still not been identified. The Ras1-cAMP pathway has been identified as a critical. If network signaling is indeed occurring during the farnesol response. it remains undetermined if it is capable of responding to farnesol under different environmental conditions. peptone. even a possible direct target for farnesol inhibition during yeast to filament inhibition by farnesol (Davis-Hanna et al..152 Signaling During the Morphological Response to Farnesol in C. 2008) showed the inability of the ras1Δ/ras1Δ mutant to respond to farnesol in aerobic conditions. Although a recent study (Davis-Hanna et al.. albicans signaling in general (Braun and Johnson. farnesol could help identify many of the different branches in these pathways. whereby many individual connections are formed between the different regulatory pathways in the cell. 2010).
it remains unknown how another factor involved. not only to study farnesol tolerance as mentioned earlier. 4). 2009).153 the existing aerobic data and determine the importance of Ras1 to the farnesol response in other environmental conditions. If this mutant is viable.. Ras2 (Zhu et al. Further studies using farnesol as a tool may decipher whether Tup1 is somehow connected to the cAMP pathway or is independently involved in the farnesol response. 2009). fits into this model (Chapters 3. More importantly. as well as ectopic expression constructs for all three genes . Although Ras2 shares poor sequence homology with Ras1 and phylogenetic analyses have suggested it belongs to a divergent group of fungal Ras proteins (Ras2 has a homolog with high sequence similarity to C. Construction of a triple efg1Δ/czf1Δ/tup1Δ mutant may prove useful. A recently characterized protein. Might Ras2 play an antagonistic response to Ras1 during farnesol signaling as well? While one new factor involved in the morphological response to farnesol. since Ras2 has an antagonistic effect on many of the physiological roles of Ras1. and TUP1 have already been constructed. Tup1. stress resistance. albicans. and cellular cAMP levels. making it an ideal candidate for testing the morphological response to farnesol. it may somehow be involved in farnesol signaling as well. dubliniensis). has a known connection to the Ras1-cAMP pathway in C.. CZF1. plays an antagonistic role with Ras1 regarding stationary phase entry. The ras2Δ/ras2Δ mutant maintains the ability to make filaments in aerobic and embedded conditions. Czf1. but to study farnesol signaling as well. it still has functional GTPase activity (Zhu et al. what would its morphology be in different growth conditions? Would this mutant be capable of responding to farnesol? Vectors that can be used to delete EFG1.
For example. . are the TUP1 mRNA levels in a czf1Δ/czf1Δ mutant background still increased during farnesol treatment? If the mRNA levels do not increase in response to farnesol. Construction and analysis of these strains can provide valuable insight into C. followup experiments designed to better understand which genes are controlled by Czf1 are desirable for the C. 1999. Lo et al. and this information could potentially provide the first data regarding the regulation of Tup1 since regulatory factors upstream of Tup1 remain unknown. 2001. a couple of which have already been obtained by the Atkin laboratory. Therefore. Other ways to test for a possible connection between Tup1 and the Ras1-cAMP pathway would be to determine whether Czf1 and/or Efg1 are responsible for the increase in TUP1 mRNA levels and/or Tup1 protein levels observed during the morphological response to farnesol. 1997. Tup1 would most likely be acting upstream of these two factors. Brown et al. but the remaining double mutant combinations (i. it is unknown what other genes are directly regulated by Czf1. 1997).e. Regardless of whether Czf1 would specifically regulate TUP1 expression. if TUP1 was ectopically expressed in the efg1Δ/efg1Δ czf1Δ/czf1Δ double mutant background and no differences in farnesol response were observed. While it has been shown to bind its own promoter and negatively regulate its expression. Moreover.. because it controls so many processes in C.. albicans morphological signaling networks. Braun and Johnson. it would suggest that Czf1 is either directly or indirectly responsible for the regulation of Tup1 during the farnesol response. all the necessary tools are available to create not only a triple mutant. albicans research field. tup1Δ/czf1Δ.154 (Bockmuhl and Ernst. efg1Δ/tup1Δ) and ectopically express each gene in the desired mutant strains. For example.
Prior to this experiment. which would have the potential to slightly alter the natural function of the protein). Either the acquisition of amino acids plays a more important role during farnesol signaling than is currently appreciated. particularly when examining the farnesol survival mechanism. it is of considerable interest to identify genes that are directly regulated by this unique transcription factor. The ChIP-Seq method would first cross-link DNA (chromatin)-protein complexes and shear the DNA into smaller pieces. ChIP (chromatin immunoprecipitation)-Seq. and the morphological response to farnesol. Sequence analyses would finally provide the location of the Czf1 binding sites in the C.155 albicans: morphogenesis. due to their known roles in stress resistance. The RLM1 and YCK2 genes are both attractive candidates for further analysis. albicans genome. and deep sequencing could subsequently be used to sequence the precipitated DNA. however. An antibody against Czf1 can be used to immunoprecipitate DNA bound with Czf1. Czf1 antibodies would ideally be raised (all protein work concerning Czf1 has utilized an HA-tagged Czf1 protein. Of note. In addition to CZF1. both without and in the presence of farnesol. white-opaque switching. farnesol tolerance. or Stp2 may participate in farnesol signaling in an undiscovered manner. identification of STP2 was a bit of a surprise. as it is the only gene identified with no direct ties to morphogenesis or stress responses. would be an ideal procedure to determine direct targets of Czf1. . the mutant screen from Chapter 4 identified 7 other genes that may play a role in the farnesol response.
Although farnesol was initially discovered as a quorum sensing molecule that only regulates morphogenesis. albicans is that farnesol affects a variety of regulatory factors within the polymorphic fungus. . Farnesol can be used as an instrument to dissect the complicated and intertwined pathways present in C. albicans.156 Final Thoughts One common theme that can be discerned from studying the farnesol response in C. albicans signaling as a whole. such as the regulation of Tup1. including pathways yet to be fully understood. and stress response signaling. This unique feature has allowed us to study the interrelatedness of some of the signaling networks in an effort to better understand the genetic pathways present in the opportunistic pathogen. Hog1 signaling. the discovery of its many other functions will hopefully allow for a more complete understanding of C.
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